Evaluation of thioamides, thiolactams and thioureas as hydrogen sulfide (H2S)donors for lowering blood pressure

Article abstract of DOI:10.1016/j.bioorg.2019.102941

Hydrogen sulfide (H₂S)is a biologically important gaseous molecule that exhibits promising protective effects against a variety of pathological processes. For example, it was recognized as a blood pressure lowering agent. Aligned with the need for easily modifiable platforms for the H₂S supply, we report here the preparation and the H₂S release kinetics from a series of structurally diversified thioamides, thiolactams and thioureas. Three different thionation methods based on the usage of a phosphorus pentasulfide and Lawesson reagent were applied to prepare the target thioamides and thiolactams. Furthermore, obtained H₂S donors were evaluated both in in vivo and in vitro studies. The kinetic parameters of the liberating H₂S was determined and compared with NaHS and GYY4137 using two different detection technics i.e.; fluorescence labeling 7-azido-4-methyl-2H-chromen-2-one and 5,5‘-dithiobis (2-nitrobenzoic acid), sulfhydryl probe, also known as the Ellman's reagent. We have proved that the amount of releasing H₂S from these compounds is controllable through structural modifications. Finally, the present study shows a hypotensive response to an intravenous administration of the developed donors in the anesthetized rats.

Full text of DOI:10.1016/j.bioorg.2019.102941

Accepted Manuscript
Evaluation of thioamides, thiolactams and thioureas as hydrogen sulfide (H S)
2
donors for lowering blood pressure
Ewelina Zaorska, Tomasz Hutsch, Marta Gawryś-Kopczyńska, Ryszard
Ostaszewski, Marcin Ufnal, Dominik Koszelewski
PII:
S0045-2068(19)30252-4
https://doi.org/10.1016/j.bioorg.2019.102941
102941
DOI:
Article Number:
Reference:
YBIOO 102941
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
14 February 2019
8 April 2019
18 April 2019
Please cite this article as: E. Zaorska, T. Hutsch, M. Gawryś-Kopczyńska, R. Ostaszewski, M. Ufnal, D.
Koszelewski, Evaluation of thioamides, thiolactams and thioureas as hydrogen sulfide (H S) donors for lowering
2
blood pressure, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.102941
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Evaluation of thioamides, thiolactams and thioureas as hydrogen sulfide
H S) donors for lowering blood pressure
(
2
[
a]
[a]
[a]
[b]
[a]
Ewelina Zaorska, Tomasz Hutsch, Marta Gawryś-Kopczyńska, Ryszard Ostaszewski, Marcin Ufnal,* and
[
b]
Dominik Koszelewski*
Affiliation and address:
[
a]
prof. M. Ufnal, E. Zaorska, M. Gawryś-Kopczyńska, Tomasz Hutsch:
Department of Experimental Physiology and Pathophysiology, Laboratory of Centre for Preclinical Research,
Medical University of Warsaw, Pawińskiego 3c, 02-106 Warsaw, Poland,
[
b]
dr. D. Koszelewski, prof. R. Ostaszewski: Institute of Organic Chemistry, Polish Academy of Sciences
Kasprzaka 44/52, 01-224 Warsaw, Poland
Corresponding authors: dominik.koszelewski@icho.edu.pl, mufnal@wum.edu.pl
Abstract: Hydrogen sulfide (H S) is a biologically important gaseous molecule that exhibits promising protective
2
effects against a variety of pathological processes. For example, it was recognized as a blood pressure lowering
agent. Aligned with the need for easily modifiable platforms for the H S supply, we report here the preparation
2
and the H S release kinetics from a series of structurally diversified thioamides, thiolactams and thioureas.
2
Three different thionation methods based on the usage of a phosphorus pentasulfide and Lawesson reagent
were applied to prepare the target thioamides and thiolactams. Furthermore, obtained H S donors were
2
evaluated both in in vivo and in vitro studies. The kinetic parameters of the liberating H S was determined and
2
compared with NaHS and GYY4137 using two different detection technics i.e.; fluorescence labeling 7-azido-4-
methyl-2H-chromen-2-one and 5,5`-dithiobis (2-nitrobenzoic acid), sulfhydryl probe, also known as the Ellman’s
reagent. We have proved that the amount of releasing H S from these compounds is controllable through
2
structural modifications. Finally, the present study shows a hypotensive response to an intravenous
administration of the developed donors in the anesthetized rats.
Keywords: hydrogen sulfide, thioamides, thiolactams, thiourea, cardiovascular
Introduction
Hydrogen sulfide (H S) at high concentrations is an offensive-smelling and toxic gas with a high
2
solubility in water. However, pioneering work of Kimura’s group revealed that at very low concentrations H2S
[
1]
plays a role of biological mediator. It is believed that H S in living organisms is responsible for the regulation
2
of several physiologically relevant processes e.g. vasorelaxation, hormone secretion, apoptosis and
multicellular events like neuro modulation or inflammatory responses.[2] Due to this reason a number of H2S
[
3]
releasing agents have been developed and validated in various biochemical studies. Among them, those
[
4]
related to H S-mediated vasodilator effects have been paid a great attention. In nature, H S is formed during
2
2

an anaerobic bacterial digestion of the organic compounds. Moreover, H S is produced in the colon by the
2
[
5]
commensal bacteria by the reduction of alimentary sulfates. Under the biological conditions hydrogen sulfide
is delivered enzymatically in metabolic pathways from L-cysteine by cystathionine β-synthase and cystathionine
γ-lyase, from D-cysteine via mercaptopyruvate sulfurtransferase (MPST) with D-amino acid oxidase, and from
[
6]
3
-mercaptopyruvate by MPST with cysteine aminotransferase. Naturally occurred and rich sources of the
[
7]
sulfur-based organic compounds is garlic. Recent studies suggest that at least some beneficial effects of garlic
are due to H S production. So far, the best characterized compound from garlic is allicin (diallyl thiosulfinate),
2
which quickly decomposes into several compounds such as diallyl sulfide (DAS), diallyl disulfide (DADS) and
diallyl trisulfide (DATS). Such as naturally H S donors may be attractive options for in vivo studies due to lack of
2
[
8-9]
toxicity, they unveil problems that accompany many synthetic donors.
However, there are some relevant
limitations to the use of these compounds: no susceptibility to chemical transformations, poor water solubility,
[
10]
creation of various byproducts after H S release.
The most common class of H S donors employed in
2
2
biological studies are the inorganic sulfide salts such as sodium hydrosulfide (NaHS) and sodium sulfide (Na S).
2
[
11-13]
Generally, sulfide salts are considered as a short-lasting H S donors as they release H S rapidly.
Such
2
2
uncontrollable and fast H S release may exert toxic actions in living systems. Moreover, these compounds
2
release H S spontaneously already in time of solution preparation. Such H S loss from aqueous solutions
2
2
making substantial problems with precisely controlled H S dose administration what finally may affect
2
biological outcome. Additionally the fact that sulfide salts are hygroscopic may be a source of error in
[
14-15]
calculations the exact H S concentration of dose to be applied to the biologic system.
Another problem
2
often raised with commercial sulfide salts, especially NaHS is that almost always they contain some amount of
[
16]
undefined impurities.
Due to these inconveniences organic molecules which act as H S agents were
2
developed and used. Thus far various types of organic donors like; dithiolthiones, thiobenzamides,
thiocarbamates, dithioesters, and thionoesters, which release H S via different mechanisms have been
2
[
17]
[18]
[19]
[20]
reported,
e.g. hydrolysis,
cellular thiol activation,
and photolysis.
The most widely studied and
characterized synthetic H2S donor is GYY4137 (morpholin-4-ium 4-methoxyphenyl(morpholino)
phosphinodithioate) complex (Figure 1). Although, GYY4137 is a water-soluble derivative of Lawessons reagent
(
LR), which releases H S via hydrolysis, the rate of H S generation firmly depends on pH and turn to be very
2
2
[
21]
slow at physiological pH. Recently, we have shown that the amount of the released H S from GYY4137 (140
2
[
22]
μM) at pH 7.4 in aqueous solution is <2 μM /2,3 μmol /90 min/13 h. Additionally, the GYY4137 suffers from
several other inconveniences. First, it is often prepared as a dichloromethane complex (ratio 3:1), which is
residual from crystallization. Dichloromethane is metabolized to carbon oxide which has biological impact
[
23]
similar to H S, therefore some effects attributed to GYY4137-derived H S may come from CO. Finally, it is
2
2
most likely that reported biological effects observed after administration of GYY4137 are not H S-dependent
2
[
24]
but can be caused by GYY4137 itself or products of its metabolism after H S release (Zheng et al., 2015).
2
Despite the number of literature data regarding H S donors only a few show the impact on the vascular system
2
provided from the in vivo studies.

Figure 1. GYY4137 dichloromethane complex.
Regardless of the significant progress in the field of H S donor chemistry, there is still a lack of
2
compounds that would meet all the requirements for ideal H S donor e.g. water-solubility and generation only
2
innocuous (if any/or not at all) byproducts. Last but not least, it should also have well-defined release
mechanism. Among of the organic H S donors, thio-analogs of amides and urea have long attracted more and
2
more attention due to their practical application as the precursors for the synthesis of various organic
[
25-28]
compounds.
They are also an important class of bioactive molecules in the field of pharmaceutical
[
29-34]
industry.
The fact that thioacetamide, one of the simplest organosulfur compound release H S by hydrolysis has
2
[
35,36]
been known for over a century.
As so far, multiple methods for thioamide and thiourea derivatives
[
37-44]
synthesis have been reported, using various reagents and diverse reaction conditions.
Encouraged by the
[
28-33]
studies revealing biological activity of thioamides and thioureas,
we envisioned those compounds as a
valuable substructures for the design of sufficient H S donors. We assumed also that the type of substituents
2
on the nitrogen atom of amino group of target compounds may provide donors with the desired properties in
controlled H S release. In this work we designed and synthesized the series of thioamides, thiolactames and
2
thioures derivatives. Then, the newly prepared compounds were evaluated for their H S-releasing properties
2
using two independent approaches i.e. fluorescence and DTNB methods. Finally, the selected compounds were
submitted to experiments aimed to evaluate their hemodynamic effect on normotensive in rats.
Results
The concept of thionation, i.e. the conversion of the carbonyl group into thiocarbonyl by exchange of
an oxygen atom for a sulfur atom is the most commonly used procedure for the preparation of organosulfur
[
45]
compounds. This transformation can be carried out with several commercially available reagents. Among
them, a phosphorus pentasulfide (P S ) and the 2,4-bis(4-methoxyphenyl)-1,3-dithiaphosphetane 2,4-disulfide,
4
10
[
46-51]
also known as the Lawesson’s reagent, are the most popular (Figure 2).
Despite the many advantages of
using P4S10 as a thionating reagent, LR is the common choice in organic synthesis. As long as a number of
reported protocols regarding LR demonstrate the utility of this reagent, only several of them concomitantly
highlighted its limitations. The major difficulties of LR application are byproducts derived from the reagent
itself, which cannot be removed by any extractive procedure and must be separated by the time consuming
chromatographic techniques. Such tedious separation protocol of the desired product from the LR byproducts
[
52,53]
can be cumbersome or even impossible, what limits the reagent utility.

Figure 2. Lawesson’s reagent (LR).
Regardless of the high popularity of the Lawesson’s reagent as a thionating agent there are several
reports refining applicability of the phosphorus pentasulfide by the addition of hexamethyldisiloxane (HMDSO).
It was shown that supplementation of P4S10 with HMDSO dramatically increases thionation rates. Moreover,
the application of P S /HMDSO system requires simple extractive workup to remove derived byproducts from
4
10
[
54,55]
the final reaction mixture.
Due to the lack of generality and potential problems with the product isolation,
both LR and P4S10 were engaged in the synthesis of the target thioamides 2 and thiolactams 3 (Scheme 1). A
comparative results obtained under different reaction conditions were shown in Table 1.
Scheme 1. Synthesis of thioamides 2a-j and thiolactams 3a-b.
Thioformamide (2a), N-(2-methoxyethyl)methanethioamide (2b) 4-thioformylmorpholine (2c) and N-
methylthioformamide (2d) were provided from the corresponding formamide derivatives 1a-j using P4S10 in
[
56]
diethyl ether with the yields ranging from 75-86%, In addition, the thionation of selected amides 1f-g was
conducted under different conditions using P4S10 alone, P S /HMDSO system and LR in three different organic
4
10
solvents; diethyl ether, DCM and THF (Table 1, entries 5-7). In all cases the application of P S /HMDSO or LR
4
10
provided corresponding thioamides 2f-g with higher yield than with P4S10 alone what remains in agreement
[
57]
with the literature data. The reaction of acetamide 1h with LR gave desired product 2h in 93% yield within
three hours (Table 1, entry 8). The influence of the used solvent on the reaction performance was negligible. As
shown in Table 1 the reaction of aromatic amides 1i and 1j (entries 9-10) with LR afforded the corresponding
target thioamides 2i and 2j in high yields up to 90%. The impact of the used solvent on the thionation was
examined. There was no evident correlation between used medium and thionation efficiency, products 2i and
2j were obtained with comparable yields (Table 1, entries 9-10). Subtly enhanced yields of the amides 2i and 2j
in THF may be explained by the higher solubility of the LR reagent in this solvent. Additionally, two different
[
58]
size-chain thiolactams were synthesized (Table 1, entries 11 and 12). Pyrrolidin-2-tione (3a) and piperidine-2-
thione (3b) were provided from the corresponding lactams; 2-pyrrolidinone (1k) and 2-piperidinone (1l), using
LR reagent in DCM (Scheme 1).
Table 1. Thionation of amides and lactams under various conditions.

o
[b]
Entry
1
Amide/Lactam
Thionation
reagent
Solvent
T [ C]
Time
[h]
Product
Yield
[%]
log P
[a]
P S
Et O
2
0→20
0→20
3
86
75
-0.33
0.02
4
10
P S
Et O
2
3
4
10
2
3
4
P S
Et O
2
0→20
0→20
3
3
80
82
0.81
0.01
4
10
P S
Et O
2
4
10
P S
Et O
0→20
60
24
24
24
75
86
87
4
10
2
P S /HMDSO
DCM
THF
0.10
-0.25
-0.23
4
10
5
6
7
LR
20
P S
Et O
0→20
60
24
24
24
70
84
80
4
10
2
P S /HMDSO
DCM
THF
4
10
LR
20
P S
Et O
2
0→20
60
24
24
24
78
90
85
4
10
P S /HMDSO
DCM
THF
4
10
LR
LR
LR
20
THF
93
89
20
20
3
-0.26
1.76
8
9
DCM
THF
24
90

DCM
THF
88
89
85
LR
0.99
10
20
24
DCM
LR
LR
DCM
DCM
20
20
2
2
93
93
-0.58
-0.02
1
1
2
1
[
a] Isolated yield. [b] Calculated using ChemBioDraw ultimate software.
The desired products were isolated in high yields up to 93% within two hours. The effect of the lactam ring size
on the reaction yield was not observed.
Thoughtful screening of the literature regarding potential H S agents provides basis that the thiourea
2
[
29-34]
could act as a H S donor.
Therefore, we designed and synthesized a series of compound based on thiourea
2
scaffold (Scheme 2).
Scheme 2. Synthesis of thiourea derivatives from the corresponding isothiocyanates.
The variously substituted thiourea derivatives 4a-e were obtained by the reaction of corresponding
[
59-61]
isothiocyanate with appropriate amine or diamine (Scheme 2).
Three different commercially available
isothiocyanate were used: methyl isothiocyanate, phenyl isothiocyanate and benzyl isothiocyanate. As shown
in the Table 2 the reaction of aromatic isothiocyanate with a structurally diversified amines possessing in their
structure hydrophilic groups afforded the corresponding thiourea derivatives 4c-e in high yields up to 87%
(Table 2, entry 3). The reactions also proceeded efficiently with both aliphatic and heterocyclic amines.
Application of methyl isothiocyanate resulted in the corresponding thiourea derivatives 4a-b with the yields up
to 92% (Table 2, entries 1 and 2). For diamines the appropriate excess of isothiocyanate (2 equiv.) was used
(Table 2, entries 2 and 4).

Table 2. Synthesis of thiourea derivatives 4a-e.
Molar ratio
[a]
[b]
Entry
R1
Amine/Diamine
Isothiocyjanate/ Solvent
amine
Product 4
Yield [%]
log P
DCM
92
89
acetone
1:1
0
.34
1
toluene
8
3
DCM
89
85
81
-
0.60
2
2:1
acetone
toluene
DCM
80
87
0
.90
3
1:1
toluene
DCM
75
80
4
2:1
1.53
toluene
1
.11
1
:1
DCM
79

5
toluene
83
[
a] Isolated yield. [b] Calculated using ChemBioDraw ultimate sofware.
The solvents such as an acetone, toluene and DCM were used. The reaction performed in DCM using
methyl isothiocyanate resulted in higher yield of the corresponding thiourea derivatives 4a and 4b than
analogous one conducted in toluene or acetone (Table 2, entries 1 and 2). In the case of isothiocyanates with
aromatic ring, toluene revealed to be the most suitable solvent (Table 2, entries 3-5). The higher yield may be
due to a higher solubility of the used reagents in given solvent. Additionally, we have developed protocol for
[
62]
the synthesis of symmetrically substituted urea derivative 5 (Scheme 3).
Scheme 3. Synthesis of 1,3-bis(2-morpholin-4-ylethyl)thiourea (5).
The 1,3-bis(2-morpholinoethyl)-thiourea (5) was obtained via reaction of 2-morpholinoethan-1-amine
with thiophosgene in DCM with 85% yield. The structures of all obtained compounds (2a-j, 3a-b, 4a-e and 5)
1
13
were confirmed by H-, CNMR spectroscopy, mass spectra analysis and elemental analysis (supplementary
materials). Spectroscopic data for the known compounds correspond with the published one. Further, we have
evaluated the potential H S-releasing capability of the synthesized compounds. Initially, we have validated the
2
newly prepared compounds toward H S liberation using recently established and commonly used procedure
2
based on DTNB probe also known as Ellman’s reagent (5,5`-dithiobis(2-nitrobenzoic acid). In general, DTNB
[
63-65]
method is used for the determination of the free sulfhydryl group concentration.
DTNB assay for 1 mM
solutions of H S donors was performed at pH 8.0 according to the protocol provided by the manufacturer
2
(
supplementary materials). As shown in Table 3 all prepared compounds liberate H S. The highest amount of
2
the released H S was observed for 2e and 2h thio-derivatives (Table 3, entries 5 and 8), while the lowest one
2
was recorded for compounds 2g, 4d and 4e (Table 3, entries 7, 16 and 17).
[
a]
Table 3. In vitro H S release evaluation from compounds 2a-j, 3a-b, 4a-f and 5 using DTNB probe.
2
Entry
Donor
2a
Conc. [μM]
254
1
2
3
4
5
2b
271
2c
407
2d
335
2e
736

6
7
8
9
2f
2g
2h
2i
180
95
434
106
115
190
267
129
102
100
85
1
0
1
2
3
4
5
6
7
8
2j
1
1
1
1
1
1
1
1
3a
3b
4a
4b
4c
4d
4e
5
95
189
[
a] 10 mM Ellman’s Reagent Solution in sodium phosphate buffer (pH
.0, 50 mM), room temperature, spectra recorded 15 minutes after
8
addition of H S donor at 412 nm. For each donors the absorbance was
2
measured at least three times. Background values were subtracted from
the sample values.
Since the DTNB operates at basic conditions which does not meet physiological one (pH 7.4) and can
affect the amount of the liberaitng H S, we turn our attention toward alternative technique which allows us to
2
measure the reactive sulfur species in physiological environment. Fortunately, the rapid progress drives the
emergence of the assay for H S detection, which possesses the biotarget sensitivity and synthetic convenience.
2
[
66]
Up to date, a variety of fluorescent probes for tracking the H S in biological samples is available. Due to the
2
fast catabolism and low stability of H S we required a probe that would react rapidly and selectively with low
2
levels of hydrogen sulfide and produce a robust fluorescent signal. In addition, the convenient probe should be
characterized by the facile synthesis with an efficient and fast protocol for purification. For these reasons a 7-
azido-4-methyl-2H-chromen-2-one (6) the non-fluorescence derivative of 7-amino-4-methylcoumarin (Scheme
[
67]
4
) have been carefully selected.
It should be emphasized that probe 6 in contrast to DTNB sensor
demonstrates high selectivity and sensitivity toward H S over other relevant reactive sulfur species in vitro, as
2
[
68]
well as identified exogenous H S in living cells. The target probe 6 was synthesized in 88% yield from m-
2
[
68]
hydroxyaniline in accordance with the previously described procedure. As reflecting diverse state-of-the-art
OFF/ON fluorescent mechanism, including the release of fluorophore triggered by reduction of an azide to the
amine was showed in Scheme 4. In our experiments, 1 mM of each donor was dissolved in a mixed
MeCN/sodium phosphate buffer solution (2:8 v/v, pH 7.4) containing probe 6 (1 mM). The changes in the
fluorescence emission spectra at 430 nm (λ = 365 nm) were recorded for one hour at room temperature.
ex

Scheme 4. Mechanism of H S detection for the probe 6.
2
Finally, the fluorescence signals were converted to H S concentrations based on a reference curve
2
obtained with a series of NaHS standard solutions. In each case H S concentrations were determined after 15
2
minutes. In order to evaluate the release properties of H S by studied agents, we carried out independent
2
verification of H S release profiles from known H S donors; NaHS and GYY4137 under the same experimental
2
2
conditions (Table 4, entries 1 and 2). Alike previously for DTNB assay, also in the fluorescence method with
probe 6 all prepared compounds showed H S-releasing capabilities. As can be expected the H S-release from all
2
2
tested compounds is lower than for NaHS. In turn amounts of H S release from each of studied donors 2a-j, 3a-
2
b, 4a-e and 5 were higher than for well recognized GYY4137. For compound 2a, 3b and 5 the highest H S-
2
releasing was observed (Table 4, entries 3, 14 and 20). The compound 2j released the smallest amounts of H S,
2
but still on the higher level than GYY 4137 (Table 4, entry 12). All other compounds were similarly found to
generate H S under studied conditions. Additionally, for arbitrary selected donors 2a, 3b and 5, fluorescence
2
measurements were performed in assay buffer with addition of rat blood plasma (20% v/v). The obtained
results were similar to those obtained in buffer alone, what implies that the intracellular environment does not
affect H S-release capability of selected donors. Differences in the H S-realising values can be associated with
2
2
the solubility of these compounds in an aqueous medium (for compound with the higher value of log P the
amount of H S released were lower). We assume that hydrophobic properties of a given compounds can
2
reduce the hydrolysis rate of the studied donors and consequently decrease the generation of H S. As can be
2
seen the H S release data for thioamide 2a and thiourea derivatives 4a-e correspond to those reordered using
2
DTNB sensor. One the other hand the higher values observed in case of using Ellman’s reagent may arise from
it varied selectivity toward sulfur species.
Table 4. In vitro H S release evaluation from compounds 2a-j, 3a-b, 4a-e and 5 determined in fluorometric
2
assay.
[a]
Fluorescence method
[b]
Entry
Donor
NaHS
GYY4137
2a
Conc. [μM]
260
1
2
3
4
5
14
228
2b
146
2c
137

6
7
8
9
2d
2e
2f
134
101
98
2g
2h
2i
96
1
0
1
2
3
4
5
6
7
8
9
0
94
1
1
1
1
1
1
1
1
1
2
92
2j
82
3a
3b
4a
4b
4c
4d
4e
5
120
182
93
107
100
100
95
191
[
a] 1 mM of probe 6 in a mixed MeCN/sodium phosphate buffer solution (2:8 v/v, pH 7.4);
a room temperature, the fluorescence response was monitored in range time from 1 to
0 minutes after addition of the H S donor. For each donors fluorescence intense was
6
2
measured three times. [b] The H S amount determined after 15 min. For calculation were
2
used the mean obtained from three measurements. Background values were subtracted
from the sample values.
Cellular nucleophiles play crucial roles in biological systems. The thiols that occur in cells in the large
amount and characterized by the strongest potent reactivity are cysteine (Cys) and reduced glutathione (GSH).
According to the literature data, release of H S from donors based on thioamide structural motif may be
2
[
69-71]
modulated by the organic thiols such as reduced glutathione or/and cysteine.
Therefore, additional
experiments were pursued in the presence of L-cysteine for selected donors 2a, 2c, 2e, 2i, 2j, 3a, 3b and 4b.
Due to the lack of selectivity of the DTNB method towards thiol groups further measurements with cysteine
were carried out with 7-azido-4-methyl-2H-chromen-2-one (6). Regardless of the literature data regarding
selectivity of the used probe 6, we have validated this compound using two different reactive sulfur species.
These studies proved that probe 6 exhibited high selectivity towards H S over cysteine and reduced glutathione
2
(
GSH) under studied conditions. As shown in Figure 3, cysteine (4 mM) successfully triggered H S release, at a
2
relatively higher level for 2e, 2i, 2j, 3a, 3b and 4b donors (Table 5, entries 3-8). While the release of H S from 2a
2
and 2c in the presence of 4 mM cysteine was lower than observed in the assay buffer alone (Table 5, entries 1-
2).

Table 5. In vitro H S release evaluation in the presence of L-Cysteine (4mM) determined in fluorometric assay.
2
[a]
Fluorescence method with probe 6
PBS/L-cysteine [4mM]
[b]
Entry
1
Donor
Conc. [μM]
2a
128
2
3
4
5
6
7
8
2c
2e
2i
95
111
96
2j
95
3a
3b
4b
136
197
123
[
a] 1 mM of probe 6 in sodium phosphate buffer (pH 7.4), 4 mM L-cysteine, room
temperature, the fluorescence response was monitored in a range time from 1 to 60
minutes after addition of the H S donor. For each donors the fluorescence intense was
2
measured three times. [b] The H S amount was determined after 15 min. For calculation
2
were used the mean obtained from three measurements. Background values were
subtracted from the sample values.
3
2
2
1
1
00
50
00
50
00
H2S donor
H2S donor + L-Cys (4 mM)
5
0
0
2
a
2c
2e
2i
2j
3a
3b
4b
H S donor
2
Figure 3. Comparison of the H S amount released from the selected H S donors (1 mM) in the presence of L-
2
2
cysteine (grey bars) and in the assay buffer alone (black bars).

Obtained results demonstrate the capability of the thioamides, thiolactams and thioureas derivatives
to release H S in complex biological systems and also show that cysteine is the regulator of this type of donors.
2
The observed differences in H S liberation between the results obtained in both used analytical methods can
2
be associated with way of sample processing and conditions of measurements which are substantially different
for the used methods. In comparison to fluorescent method, DTNB method requires alkaline conditions (pH
8
.0) The above factors can lead to falsely elevated or decreased H S amounts in samples and may be the cause
2
of the observed differences in the results obtained.
Haemodynamic effects of investigated H S donors.
2
Mean arterial blood pressure (MABP) and heart rate (HR) were measured at baseline and after
intravenous administration of a vehicle and newly synthesized H S donors. The compounds 2a, 2c, 2d, 2j, 3a, 4b
2
and 5 were selected as representatives due to their adequate solubility in the vehicle and proved H S release at
2
physiological pH (the fluorescence method). Additionally, we conducted experiments in which hemodynamic
effects of thiourea were investigated. The impact of selected H S donors on MABP and HR in the normotensive
2
rats are shown in Figures 4-8. There were no significant differences in MABP and HR at baseline between the
experimental series. The vehicle did not affect MABP and HR. All investigated H S donors produced
2
hemodynamic effects, however, there were differences in the onset and the size of the responses. Specifically,
the greatest decrease in MABP was present after administration of 2a whereas the lowest drop in MABP was
present after administration of 3a. Simultaneously, a significant reduction in the heart rate was observed. It is
important to mention that similar effect has not been observed for the parent lactam 2-pyrrolidinon under
similar physiological conditions (Supplementary Materials). The H S donors 2a, 2c, 2d produced immediate
2
response. In contrast thiourea and the donor 2j produced decrease in MABP 45 min and 30 min after the
administration, respectively. In general, decrease in MABP was accompanied by a decrease in HR for H2S
donors 2a, 2c, 2d, 2j and 3a. However, donor 5 produced a decrease in MABP which was associated with an
unstable increase in HR.

A
B
Figure 4. Change in (A) mean arterial blood pressure (MABP, mmHg) and (B) heart rate (HR, beats/min) after the
intravenous administration of vehicle (20% v/v of DMSO in saline, n=5) or H S donors [2a (n=5), 2c (n=5) and 2d (n=5) at
2
a dose of 0.8 mmol/kg/BW] in normotensive Sprague Dawley rats.
†
#
&
*
p<0.05 – vs. baseline, p<0.05 – vehicle vs. 2a series., v p<0.05 – vehicle vs. 2c series, p<0.05 – vehicle vs. 2d series
$
§
‡
p<0.05 – 2a vs. 2c series, p<0.05 – 2a vs. 2d series. p<0.05 - 2c vs. 2d series.
A
B
Figure 5. Change in (A) mean arterial blood pressure (MABP, mmHg) and (B) heart rate (HR, beats/min) after the
intravenous administration of vehicle (20% v/v of DMSO in saline, n=5) or H S donor [3a (n=5) at a dose of 0.8
2
mmol/kg/BW] in normotensive Sprague Dawley rats.
#
*
p<0.05 – vs. baseline, p<0.05 – vehicle vs. 3a series.

A
B
Figure 6. Change in (A) mean arterial blood pressure (MABP, mmHg) and (B) heart rate (HR, beats/min) after the
intravenous administration of vehicle (20% v/v of DMSO in saline, n=5) or H S donor [thiourea (n=5) at a dose of 0.8
2
mmol/kg/BW] in normotensive Sprague Dawley rats.
#
*
p<0.05 – vs. baseline, p<0.05 – vehicle vs. thiourea series.
A
B
Figure 7. Change in (A) mean arterial blood pressure (MABP, mmHg) and (B) heart rate (HR, beats/min) after the
intravenous administration of vehicle (20% v/v of DMSO in saline, n=5) or H S donor [2j (n=5) at a dose of 0.8
2
mmol/kg/BW] in normotensive Sprague Dawley rats.
#
*
p<0.05 – vs. baseline, p<0.05 – vehicle vs. 2j series.

A
B
Figure 8. Change in (A) mean arterial blood pressure (MABP, mmHg) and (B) heart rate (HR, beats/min) after the
intravenous administration of vehicle (20% v/v of DMSO in saline, n=5) or H S donors [4b (n=5) and 5 (n=5) at a dose of
2
0
*
.8 mmol/kg/BW] in normotensive Sprague Dawley rats.
#
†
$
p<0.05 – vs. baseline, p<0.05 – vehicle vs. 4b series, p<0.05 – vehicle vs. 5 series, p<0.05 – 4b vs. 5 series.
Conclusions
By preparing and directly comparing thioamide, thiolactam and thiourea donor platforms, we
demonstrate access to a wide array of structural motifs that function as controllable hydrolysis-based
H S donors. The liberating H S ability of these compounds was determined and compared with NaHS
2
2
and GYY4137. Obtained results proved that controllable H S release can be achieved by structural
2
modifications of the donors. We also found that thiolactam pyrrolidin-2-thione (3a) reduces heart rate
with no significant effect on blood pressure in normotensive Sprague Dawley rats. Further studies
evaluating mechanisms of the thiolactams action on the circulatory system are needed. Altogether
obtained results highlighted the potential of investigated compounds as the H S-donors for basic
2
studies and for the rational design of pharmacotherapeutic agents to treat cardiovascular disorders.
Materials and Methods
General methods. All the chemicals were obtained from commercial sources and the solvents were of
1
13
analytical grade. H- and CNMR spectra were recorded in CDCl , DMSO-d or acetone-d . Chemical shifts are
3
6
expressed in parts per million. The coupling constants (J) are given in hertz (Hz). The elemental analyses were
performed on CHN Perkin-Elmer 240 apparatus. TLC analyses were done on Kieselgel 60 F254 aluminum sheets.
Column chromatography was performed on Merck silica gel 60/230-400 mesh. SiliCycle 60 F254 silica gel sheets
were used for analytical thin layer chromatography (TLC) and visualized by fluorescence quenching under UV

light. Commercial reagents for quantitating sulfhydryl groups by DTNB method were purchased from Thermo
Fisher Scientific (Waltham, Mass., USA). Buffer reagents were purchased from Sigma Aldrich (Saint Louis, Mo.,
USA) and were used without purification. All spectroscopic measurements were performed in 0.1 mM sodium
phosphate buffer (pH 7.4) or 0.1 M sodium phosphate buffer (pH 8.0). UV/Vis spectra were recorded at
ambient temperature using a U-1900 spectrophotometer (Hitachi, Chiyoda, Tokyo, Japan) and quartz cuvettes.
Fluorescence spectra were recorded at 20 ºC in quartz cuvettes using a F7000 spectrofluorometer (Hitachi).
General procedure for thionation reaction using P S (Method A). To a cooled (0-5°C) solution of appropriate
4
10
amide 1 (1.0 mmol) in diethyl ether (3.0 mL) was added tetraphosporus decasulfide (0.25 mmol) in small
portions. The reaction mixture was allowed to reach the ambient temperature and stirred for appropriate time.
After the completion, the reaction mixture was filtered and concentrated under vacuum. To the resulting
mixture was added silica gel, concentrated in vacuum and purified by silica gel column chromatography using
hexanes/ethyl acetate as an eluent to give the title compound 2.
N,N'-((ethane-1,2-di-yl-bis(oxy))bis(ethane-2,1-diyl))dimethanethio-amide (2f). The reaction was carried out
according to the general Method A with N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))-di-formamide 1f.
1
The compound 2f was obtained with 70% yield (165 mg, 0.7 mmol). H NMR (400 MHz, DMSO) δ 10.11 (br s,
1
3
1
H), 9.35-9.13 (m, 1H), 3.70-3.63 (m, 2H), 3.58 (t, J = 5.2 Hz, 2H), 3.54-3.41 (m, 5H), 3.31 (s, 3H); C NMR (100
MHz, DMSO) δ 192.07, 189.06, 70.03, 70.02, 70.01, 69.35, 67.72, 67.66, 48.72, 43.29; Element. Anal. calcd. for
C H N O S : C: 40.66, H: 6.82, N: 11.85; found C: 40.49, H: 6.75, N: 11.81; HRMS (ESI) calcd for C H N O S Na
8
16
2
2
2
8
16
2
2 2
+
(
M+Na) 259.0551; found 259.0549.
N-(2-morpholinoethyl)methanethioamide (2g). The reaction was carried out according to the general Method
A with N-(2-morpholinoethyl)formamide 1g. The compound 2g was obtained with 78% yield (136 mg, 0.78
1
mmol). H NMR (400 MHz, CDCl ) δ 10.11 (s, 1H), 9.21 (d, J = 6.4 Hz, 1H), 3.55 (q, J = 7.8, 6.2 Hz, 2H), 3.30 (s,
3
13
6
H), 2.71-2.23 (m, 4H); C NMR (100 MHz, CDCl ) δ 188.86, 66.98, 66.40, 55.17, 53.30, 38.96; Element. Anal.
3
calcd. for C H N OS: C: 48.25, H: 8.10, N: 16.08; found C: 48.05, H: 8.73, N: 15.92; HRMS (ESI) calcd. for
7
14
2
+
C H N OSNa (M+Na) 197.0725; found 197.0722.
7
14 2
General procedure for thionation using P S /HMDSO (Method B). Amide 1 (1.0 mmol), P S (0.25 mmol),
4
10
4 10
DCM (1.0 mL) and HMDSO (1.67 mmol) were combined, refluxed and stirred 24 h. After this time the reaction
mixture was cooled to 0°C and aqueous K CO solution (1.26 mL of 5.3 M/ mmol of P S taken) was added.
2
3
4 10
Then to the reaction mixture acetone (equal to one-half of the reaction solvent) and water (1 mL/mmol of P4S10
taken) were added. The reaction mixture was stirred vigorously for 30 min at 0°C. After this time water and an
extraction solvent were added, the layers were separated, and the organic phase was washed with aqueous
solution of K CO , water, and brine. The organic extract was dried over MgSO and evaporated, and the crude
2
3
4
product was purified by column chromatography using hexanes/ethyl acetate as an eluent to give product 2.
N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))dimethanethio-amide (2f). The reaction was carried out
according to the general Method B with N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diformamide (1f).

1
13
The compound 2f was obtained with 84% yield (199 mg, 0.84 mmol). H- and CNMR data were similar to
those obtained previously.
N-(2-morpholinoethyl)methanethioamide (2g). The reaction was carried out according to the general Method
B with N-(2-morpholinoethyl)formamide (1g). The compound 2g was obtained with 90% yield (157 mg, 0.9
1
13
mmol). H- and CNMR data were similar to those obtained previously.
General procedure for thionations with LR (Method C). To a THF solution of appropriate amide 1 (1.0 mmol)
was added Lawesson’s reagent (0.5 mmol) at room temperature and the mixture was stirred for 24 h. To the
resulting mixture silica gel was added and concentrated in vacuum. The residue was purified by column
chromatography using hexanes/ethyl acetate as an eluent to give product 2.
N,N'-((ethane-1,2-diyl-bis(oxy))bis(ethane-2,1-diyl))dimethanethio-amide (2f). The reaction was carried out
according to the general method A with N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diformamide (1f).
1
13
The compound 2f was obtained with 80% yield (189 mg, 0.8 mmol). H- and CNMR data were similar to those
obtained previously.
N-(2-morpholinoethyl)methanethioamide (2g). The reaction was carried out according to the general Method
A with N-(2-morpholinoethyl)formamide (1g). The compound 2g was obtained with 85% yield (148 mg, 0.85
1
13
mmol). H- and CNMR data were similar to those obtained previously.
General procedure for preparation of thiourea derivatives 3. Method D. The phenyl isothiocyanate (135 mg
1
.0 mmol) was dissolved in toluene (5 mL) and then appropriate amine or diamine was added (1.0 or 2.0 mmol,
respectively). The reaction mixture was stirred for 24 h. The precipitate was collected by filtration, washed with
toluene, concentrated in vacuum. The residue was purified by column chromatography using hexanes/ethyl
acetate as an eluent to afford 4d.
1
,1'-(azane-di-yl-bis(ethane-2,1-di-yl))bis(3-phenylthiourea) (4d). The reaction was carried out according to
the general Method D with 1-(2-aminoethyl)ethane-1,2-diamine. The compound 4d was obtained with 78%
1
yield (291 mg, 0.78 mmol). H NMR (400 MHz, CDCl ) δ 8.33 (s, 1H), 7.48-7.19 (m, 10H), 4.06 (s, 1H), 3.90 (d, J =
3
7
5
.4 Hz, 1H), 3.53 (d, J = 6.8 Hz, 6H); Element. Anal. calcd. for C H N S : C: 57.88, H: 6.21, N: 18.75; found C:
18 23 5 2
+
7.71, H: 6.03, N: 18.57; HRMS (ESI) calcd for C H N S Na (M+Na) 396.1293; found 396.1291.
18 23 5 2
General procedure for preparation of thiourea derivatives 3 in DCM. Method E. A solution of appropriate
°
amine or diamine (1.0 or 2.0 mmol, respectively) in dichloromethane (10 mL) was cooled to 0 C followed by the
addition of triethylamine (3.0 mmol). Corresponding phenyl isothiocyanate (1.0 mmol) was added to the
reaction mixture and stirred at room temperature for 12 h (monitored by TLC for completion). The reaction
mixture was washed with water (3 x 10 mL) followed by brine (3 x 10 mL). Organic layer was dried over MgSO4
and concentrated under reduced pressure. The crude product was purified by silica gel column
chromatography using hexanes/ethyl acetate as an eluent to give 4d.

1
,1'-(azanediylbis(ethane-2,1-diyl))bis(3-phenylthiourea) (4d). The reaction was carried out according to the
general Method E with 1-(2-aminoethyl)ethane-1,2-diamine. The compound 4d was obtained with 75% yield
280 mg, 0.75 mmol).
(
General procedure for H S detection by fluorescence method in sodium phosphate buffer. The solutions of
2
probe 6 (1 mM) and tested H S donor (1 mM) were prepared in sodium phosphate buffer at pH 7.4 (20 %
2
CH CN). For the assay, appropriate donor was added to test tube / sample cuvette solution containing probe 6.
3
The total volume of the solution being measured was 2000 μL. The final concentration of probe 6 and donor in
test tube / sample cuvette were 1 mM. The fluorescence response was monitored over time. Emission spectra
were collected between 350 nm and 550 nm. For all measurements, the excitation wavelength was 365 nm.
The measurement points covered the time range from 1 to 60 min after addition of donor. For each donors
fluorescence intense was measured three times and the results were averaged.
General procedure for H S detection by fluorescence method in the presence of cysteine. The solution of
2
probe 6 (1 mM) and tested H S donor (1 mM) were prepared in sodium phosphate buffer containing 20%
2
CH CN (pH= 7.4). In these experiments appropriate donor (1 mM) was added directly to the test tubes/sample
3
cuvette solution containing probe 6 and cysteine. The final concentration of probe 6 and donor were 1 mM,
while concentration of cysteine was 4 mM. The total volume of the solution being measured was 2000 μL.
Emission spectra were collected between 350 nm and 550 nm. The fluorescence response of probe 6 was
monitored with λ =365 nm. The measurement points covered the time range from 1 to 60 min after addition
ex
of donor. For each donor fluorescence intense was measured three times and the results were averaged.
General procedure for H S detection using the Ellman’s test. For each sample to be tested were prepared test
2
tube/sample cuvette containing 50 μL of Ellman’s reagent solution and 2.5 mL of reaction buffer (Ellman’s
reagent solution and reaction buffer were prepared according to the procedure/ manual attached by Thermo
Fisher Scientific (Catalog number: 22582). Then to the separate test tubes/sample cuvette prepared in step 1
was added 250 μL of donor solution (1 mM) (for reference cuvette was added 250 μL of reaction buffer,
respectively). 1 mM Solution of donor was prepared using reaction buffer as a solvent. Then test tubes/ sample
cuvette were mixed and incubated at room temperature for 15 minutes. After this time was measured
absorbance of each sample at 412 nm. For each donors absorbance was measured three times and the results
were averaged. Concentration of sulfhydryl groups in each samples was calculate from the molar extinction
-
1
-1
coefficient of the TNB anion (ε = 14.150 M cm ) according to the procedure/manual attached by Thermo
Fisher Scientific (Catalog number: 22582).
Animal experiments. The experiments were performed according to Directive 2010/63 EU on the protection of
nd
animals used for scientific purposes and approved by the 2 Local Bioethical Committee in Warsaw
(permission number: WAW2/127/2018). The animals were obtained from the Animal Breeding Department of
the Medical University of Warsaw and housed in the Central Laboratory of Experimental Animals in group cages
with access to standard laboratory chow and water, ad libitum. The rats were maintained in a temperature-
and humidity- controlled room with a 12/12-hour light-dark cycle. The studies were performed on male, 18-20-

weeks-old, normotensive Sprague Dawley rats (SD). All measurements were performed under general
anaesthesia with urethane (Sigma-Aldrich) given intraperitoneally (IP) at a dose of 1.5 g/kg of body weight
(BW). Before the measurements rats were implanted with a venous catheter and an arterial catheter. The
arterial catheter was connected to the Biopac MP 150 (Biopac Systems, Goleta, USA) for haemodynamic
recordings. The venous catheter was used for administration of investigated compounds.
Reagents/Drugs. The following drugs were used: compound 2a, 2c, 2d, 2j, 3a, 4b, 5 and thiourea used as a H2S
donor. The solution of H S donor was prepared in 0.9% saline containing 20% v/v dimethyl sulfoxide (DMSO).
2
Haemodynamic measurements. The measurements started 60 minutes after the induction of anaesthesia and
stabilization of haemodynamic parameters. Arterial blood pressure was recorded 20 min at baseline (before
treatment) and 60 min after intravenous infusions of either 0.3 ml of the vehicle (20% v/v of DMSO (dimethyl
sulfoxide) in saline (aqueous 0.9% NaCl)) or H S donors at a dose of 0.8 mmol/kg BW (n=5).
2
Data analysis and statistics. The data are expressed as means + SEM (standard error). Mean arterial blood
pressure (MABP) and heart rate (HR) were calculated on the arterial blood pressure tracing by AcqKnowledge
4
.3.1 Biopac software (Biopac Systems, Goleta, USA). For the evaluation of MABP and HR response within the
series, baseline recordings were compared with recordings after administration of evaluated compounds by
means of the analysis of variance (ANOVA) for repeated measures. Comparisons between the treatments were
performed by ANOVA. If ANOVA showed a significant difference, the post-hoc Tukey’s test was performed. A
value of two-sided p<0.05 was considered significant. Analyses were conducted using STATISTICA 13.0.
Acknowledgements
This study was supported by The National Science Centre of Poland under grant No 2016/22/E/NZ5/00647.
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Highlights:
Thioamides, thiolactams and thioureas (TTT) may serve as hydrogen sulfide donors.
Hydrogen sulfide release rate is controllable via structural modifications of TTT.
TTT lower arterial blood pressure in rats.
Graphical abstract: