Arylthioamides as H2S donors: L-cysteine-activated releasing properties and vascular effects in vitro and in vivo

Article abstract of DOI:10.1021/ml400239a

A small library of arylthioamides 1-12 was easily synthesized, and their H₂S-releasing properties were evaluated both in the absence or in the presence of an organic thiol such as l-cysteine. A number of arylthioamides (1-3 and 7) showed a slow and l-cysteine-dependent H₂S-releasing mechanism, similar to that exhibited by the reference slow H₂S- releasing agents, such as diallyl disulfide (DADS) and the phosphinodithioate derivative GYY 4137. Compound 1 strongly abolished the noradrenaline-induced vasoconstriction in isolated rat aortic rings and hyperpolarized the membranes of human vascular smooth muscle cells in a concentration-dependent fashion. Finally, a significant reduction of the systolic blood pressure of anesthetized normotensive rats was observed after its oral administration. Altogether these results highlighted the potential of arylthioamides 1-3 and 7 as H ₂S-donors for basic studies, and for the rational design/development of promising pharmacotherapeutic agents to treat cardiovascular diseases.

Full text of DOI:10.1021/ml400239a

Letter
pubs.acs.org/acsmedchemlett
Arylthioamides as H2S Donors: L‑Cysteine-Activated Releasing
Properties and Vascular Effects in Vitro and in Vivo
Alma Martelli, Lara Testai, Valentina Citi, Alice Marino, Isabella Pugliesi, Elisabetta Barresi, Giulia Nesi,
Simona Rapposelli, Sabrina Taliani,* Federico Da Settimo, Maria C. Breschi, and Vincenzo Calderone*
̀
Dipartimento di Farmacia, Universita di Pisa, via Bonanno, 6, I-56126 Pisa, Italy
S
* Supporting Information
ABSTRACT: A small library of arylthioamides 1−12 was easily synthesized, and their H2S-releasing properties were evaluated
both in the absence or in the presence of an organic thiol such as L-cysteine. A number of arylthioamides (1−3 and 7) showed a
slow and L-cysteine-dependent H2S-releasing mechanism, similar to that exhibited by the reference slow H2S-releasing agents,
such as diallyl disulfide (DADS) and the phosphinodithioate derivative GYY 4137. Compound 1 strongly abolished the
noradrenaline-induced vasoconstriction in isolated rat aortic rings and hyperpolarized the membranes of human vascular smooth
muscle cells in a concentration-dependent fashion. Finally, a significant reduction of the systolic blood pressure of anesthetized
normotensive rats was observed after its oral administration. Altogether these results highlighted the potential of arylthioamides
1−3 and 7 as H2S-donors for basic studies, and for the rational design/development of promising pharmacotherapeutic agents to
treat cardiovascular diseases.
KEYWORDS: Hydrogen sulphide, H2S-releasing drugs, cardiovascular system, hypertension, potassium channels, hybrid drugs,
vascular smooth muscle, thioamide
exhibiting the pharmacodynamic profile of H2S-releasing agents
ydrogen sulfide (H2S) is emerging as a hot topic in the
Hfield of drug discovery. Indeed, it is recognized as an may be viewed as both powerful tools for basic studies and new
potential pharmacotherapeutic agents for treatment of many
cardiovascular diseases.
important physiological gasotransmitter in mammalian body.
Particularly, it plays a key role in regulating the cardiovascular
homeostasis by acting as a vasodilator; this effect is mainly due
to the activation of vascular ATP-sensitive (KATP) and voltage-
gated (Kv7) potassium channels.1−4 In mice models, blunted
levels of H2S in blood, heart, and aorta are associated with
increased blood pressure and decreased endothelium-mediated
vasorelaxant effects.5 In addition, in experimental models of
hypertension, it has been evidenced that exogenous H2S can
effectively prevent the progression of the pathology6 and
decrease the blood pressure values.3 Therefore, impaired
production of endogenous H2S is likely to contribute to the
pathogenesis of hypertension, highlighting the great potential
usefulness of the pharmacological modulation of this
gasotransmitter.7,8 As the administration of gaseous H2S is
greatly limited by the risk of a poor posology control and
overdose, the use of appropriate chemicals, behaving as H2S-
releasing agents, appears to be safer. Thus, compounds
NaHS is the prototypical example of a H2S-generating agent:
it is a rapid H2S donor and the most widely used H2S donor for
experimental purposes. However, this salt is not suitable for
clinical purposes, as the quick release of H2S may cause adverse
effects, such as a rapid and excessive lowering of blood
pressure.9 For a safer and effective pharmacological admin-
istration, ideal H2S donors should generate H2S with slower
releasing rates.10,11 Organic polysulphides of garlic, such as
diallyl disulfide (DADS, Chart 1), act as H2S-releasing
compounds, with a relatively slow mechanism, requiring the
presence of reduced glutathione.12 Other examples of original
synthetic H2S-releasing agents have been described in the
Received: December 21, 2012
Accepted: August 8, 2013
© XXXX American Chemical Society
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Chart 1. Structures of Known and Newly Synthesized H2S-
Releasing Agents 1−12
Scheme 1. Synthesis of 1−7, 13, and 14
Scheme 2. Synthesis of 8−12
literature, including a number of aminothiol derivatives,13 and
the phosphinodithioate derivative GYY4137 (morpholin-4-ium
4-methoxyphenylmorpholinophosphinodithioate, Chart 1).14
Furthermore, some H2S-releasing chemical moieties, such as
the dithiolethione,15−18 the thioamide, and the isothiocyanate,
have been used for synthesizing multifunctional drugs.19,20
In this work, a small library of arylthioamides (compounds
1−12, Chart 1) was easily prepared through suitable synthetic
routes and evaluated for their H2S-releasing properties. The p-
hydroxybenzothioamide 119,20 was selected as lead compound
to build the library by inserting a number of modifications on
the aromatic moiety: (i) introduction of different groups (Cl, F,
CF3, NO2, and CH3) at the 2- and/or 5-position of the phenyl
ring (2−6); (ii) replacement of the 4-hydroxy group with an
amino moiety (7); and (iii) replacement of the phenyl ring with
electron poor or electron rich heterocycles such as pyridine (8)
and piperazine (9), or furane (10), thiophene (11), and indole
(12), respectively (Chart 1). Finally, compound 1 was
submitted to further experimental protocols aimed to evaluate
the vasorelaxing effects on rat aortic rings, the membrane
hyperpolarizing activity on human vascular smooth muscle
(VSM) cells and the effects on the blood pressure of
normotensive rats.
The target compounds 1−4 and 7 were easily obtained by
reaction of the appropriate benzonitrile and P4S10 in ethanol at
70 °C for 10 h (Scheme 1). The same procedure allowed the
preparation of the methoxy-substituted intermediates 13 and
14, which were demethylated with BBr3 in dry dichloromethane
to yield derivatives 5 and 6 (Scheme 1).
The heterocyclic compounds 8−12 were prepared from the
corresponding amides by treatment with Lawesson’s reagent in
chlorobenzene at 130 °C for 12 h and in dry THF at room
temperature for 12 h, for 11 and 8−10, 12, respectively
(Scheme 2). All the products were finally purified by
recrystallization from toluene or flash chromatography
(Supporting Information).
process. In addition, since the release of H2S from DADS is
reported to be a process, which requires the presence of organic
thiols such as reduced glutathione,12 the experiments were also
performed in the presence of 4 mM L-cysteine. NaHS, DADS,
and GYY4137 were also assayed for comparison purposes. As
expected, the addition of 1 mM NaHS at pH 7.4, both in the
presence or in the absence of L-cysteine, was followed by an
immediate formation of high concentration of H2S (about 200
μM). The concentration of H2S showed a rapid fall (by about
40%) in the first two minutes after the addition of NaHS,
followed by a slower and apparently constant decline in the
remaining time (data not shown).
In order to obtain a satisfactory description of the kinetics of
those compounds generating H2S with a slow and progressive
process, the parameters of Cmax (maximal concentration of H2S
at the steady state) and t1/2 (time required to reach a
concentration = 50% of Cmax) were extrapolated from the
curves H2S-release vs time. Figure 1 reports the curves H2S-
release vs time for DADS, GYY4137, and 1, taken as
representative of the thioamide series. The values of Cmax and
t1/2 of the tested compounds are listed in Table 1.
The incubation of DADS and GYY4137 in the assay buffer
led to a negligible formation of H2S, while in the presence of L-
cysteine, a slow and significant release of H2S was observed.
Analogously, in the assay buffer alone, p-hydroxybenzothioa-
mide 1 did not show significant H2S-releasing properties. In
contrast, 1 exhibited an L-cysteine-dependent release of H2S
(Figure 1 and Table 1). In order to evaluate the possible
influence of other organic thiols, the H2S-releasing properties of
1 were also tested in the presence of reduced glutathion
(GHS). Like L-cysteine, even GSH triggered a significant, but
slower, release of H2S from 1 (Figure 1 and Table 1).
In order to characterize the potential H2S-releasing proper-
ties of arylthioamides 1−12, all the compounds were added at 1
mM concentration to the assay buffer (pH 7.4, 35−37 °C). 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
As concerns the hydroxybenzothioamide derivatives 2−6
(Table 1), the incubation of 2 (2-Cl), 3 (2-F), and 6 [3,5-
(CH3)2] led to a L-cysteine-dependent release of H2S. The Cmax
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ACS Medicinal Chemistry Letters
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Figure 1. Curves describe the increase of H2S concentration, with respect to time, following the incubation of DADS, GYY 4137, and 1 in the assay
buffer, in the absence (white squares) or in the presence of L-cysteine (black squares) or glutathion (black triangles). H2S was recorded by
amperometry; the vertical bars indicate the SEM.
be due to an easy and spontaneous hydrolysis of the H2S-
releasing moiety.
Table 1. Parameters of Cmax and t1/2 Relative to the H2S-
Releasing Effects Exhibited by the Arylthioamides 1-12 and
Reference Drugs, after Their Incubation in the Assay Buffer
at Physiological pH and Temperature, in the Absence or in
Presence (+L-Cysteine) of 4 mM L-Cysteine; nc = Not
Calculated
This first phase of the experimental work led to identify a
number of H2S-releasing arylthioamides (1−3 and 7) that
showed an L-cysteine-dependent mechanism. Such a profile has
been suggested to be convenient for the development of a
potential drug, as this feature may provide a selective H2S-
release in a biological environment (i.e., in the presence of
endogenous organic thiols such as L-cysteine, reduced
glutathion, etc.).
Compound 1 was selected for further pharmacological
studies on the basis of the following issues: (i) 1 showed a
thiol-dependent H2S-releasing profile; (ii) although it has been
already reported as a H2S-releasing side-chain in naproxen
analogues endowed with reduced toxicity,19,20 its H2S-mediated
cardiovascular effects have not been yet specifically investigated.
First, the influence of 1 (or the relative vehicle) on the
vasoconstrictor effects of noradrenaline (NA) was tested in
isolated rat aortic rings and compared with that of NaHS
(Figure 2).11 NA produced a strong contractile response, with
assay buffer
Cmax (μM) t1/2 (min)
assay buffer + L-cysteine
compound
Cmax (μM)
t1/2 (min)
1
<2
nc
3.6 0.6
10.8 2.6
15.2 3.9
<2
2.1 0.2
2.6 0.8
2.2 0.7
nc
2
<2
nc
3
<2
nc
4
<2
nc
5
<2
nc
<2
nc
6
6.1 0.3
<2
5.0 0.3
nc
11.6 0.3
6.0 2.8
16.1 5.0
21.4 1.0
10.6 0.7
5.7 0.5
15.9 1.6
19.4 5.5
10.3 2.6
4.1 0.4
4.3 1.0
3.1 0.2
6.4 0.6
5.8 0.4
1.5 0.4
9.2 0.7
1.5 0.3
2.5 0.8
7
8
3.1 1.1
14.8 0.5
7.9 0.7
4.7 0.4
14.4 0.6
<2
3.1 0.6
13.7 1.7
7.5 0.5
1.8 0.4
11.7 1.0
nc
9
10
11
12
DADS
GYY4137
<2
nc
parameters of 2, 3 and 6 were higher than that exhibited by 1
and were comparable to those of the reference drugs DADS
and GYY4137. Moreover, it should be noted that in the
absence of L-cysteine, the H2S-release from 2 and 3 was
negligible, whereas the release from 6 was lower than that
observed in the presence of the aminoacid, although it was still
significant. Compounds 4 and 5, bearing strong electron-
withdrawing groups, such as 2-CF3 and 2-NO2, respectively,
showed L-cysteine-dependent H2S-releasing properties signifi-
cantly lower with respect to the parent compound 1. Finally,
the replacement of the 4-OH group of 1 with a primary amino
moiety (see compound 7) led to a slight, albeit not significant,
improvement of the H2S-release.
Mixed results are associated with the substitution of the
phenyl ring with electron poor or electron rich heterocycles
(8−12). In particular, the pyridylthioamide 8 (electron poor)
and the thienylthioamide 11 (electron rich) both exhibited a
modest but significant generation of H2S in the absence of L-
cysteine; whereas, in the presence of the aminoacid, compound
8 showed a dramatically increased Cmax, a feature not possessed
by compound 11. The heterocyclic derivatives 9 (electron
poor), 10, and 12 (electron rich) exhibited high and L-cysteine-
independent H2S-releasing effects. The profile of L-cysteine-
independent H2S donor of deivatives 9, 10, and 12 is likely to
Figure 2. Concentration−response curves to NA, obtained on
endothelium-denuded rat aortic rings preincubated with NaHS
(squares), 1 (diamonds), or the corresponding vehicle (spots). The
effects are expressed as a % of the contractile responses previously
induced by the administration of 60 mM KCl. The vertical bars
indicate the SEM.
an Emax of 128 7 and a potency value (pEC50) of 7.76 0.08.
In the presence of 1 mM NaHS, the vasoconstrictor effects of
NA were strongly inhibited (Emax 44 3; pEC50 6.98 0.09).
It should be highlighted that, at pH 7.4 and 37 °C, 1 mM NaHS
is expected to readily produce an initial and transient
concentration of H2S of about 200 μM, due to the protonation
of the hydrosulphide anion.21 Such a theoretical hypothesis is
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ACS Medicinal Chemistry Letters
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well confirmed by the above experimental data. Interestingly,
the pretreatment of the aortic rings with compound 1 (1 mM)
almost completely abolished the ability of NA to evoke any
vasoconstriction (Figure 2).
Noteworthy, the previous amperometric measurements
indicated that 1 ensured a long-lasting release of relatively
low concentrations of H2S, requiring the presence of L-cysteine
or glutathion; however, in this experimental setup, exogenous L-
cysteine or glutathion were not added. Therefore, the above
experimental result suggests that (a) the amounts of organic
thiols endogenously present in the biological sample (i.e., the
aortic rings) can ensure the release of H2S from 1; (b) a
prolonged presence of relatively low concentrations of H2S
seems to be highly effective in modulating the activity of
vasoconstrictor agents, such as NA. In order to confirm the
above hypotheses, compound 1 was also incubated in Tyrode
buffer (i.e., in the same conditions of the functional
experiments), in the presence or in the absence of rat aortic
tissue. Neither L-cysteine nor glutathion were exogenously
added. The formation of H2S was continuously evaluated by
amperometry. In the presence of the vascular tissue (rat aortic
rings), the addition of compound 1 led to a significant time-
dependent formation of H2S. Fifteen minutes after the
incubation, the concentration of H2S in the Tyrode buffer
was 0.24 0.05 μM (Figure 3). This evidence indicates that
Figure 4. Hyperpolarizing effects induced by LCRK, NaHS, and
increasing concentrations of 1 in HASMCs. Data are calculated as
changes in relative fluorescence and then expressed as a % of the
maximal effect induced by LCRK. The vertical bars indicate the SEM.
and 4), showing a 79 22% hyperpolarizing effect (expressed
as a % of the effect produced by LCRK at 10 μM) at the highest
concentration (1 mM). As already observed in rat aortic rings,
these results indicate that the release of H2S from 1 can be
promoted by the organic thiols endogenously present in the
HASMCs and that the long-lasting presence of relatively low
concentrations of H2S is more effective than a high and
transient peak concentration.
Finally, the influence of 1 and NaHS on the blood pressure
of normotensive rats was tested. The anesthetized normoten-
sive rats showed a basal level of systolic pressure of 134
2
mmHg. After oral administration of the vehicle, no significant
change of the systolic blood pressure was observed (102 1%
of the basal levels), whereas the systolic blood pressure of the
animals was significantly reduced after oral administration of
0.1 mg/kg NaHS (91 1% of the basal levels) and equimolar
doses of 1 (89 1% of the basal levels); Figure 5.
Figure 3. Curves describe the increase of H2S concentration, with
respect to time, following the incubation of 1 in Tyrode buffer, in the
absence (white squares) or in the presence of aortic tissue (black
squares). H2S was recorded by amperometry; the vertical bars indicate
the SEM.
compound 1 is effectively transformed into H2S, by means of
biomolecules (probably, organic thiols) endogenously present
in the vascular tissue; in particular, effective H2S concentrations
are likely to be produced in the intracellular compartment (i.e.,
where the levels of endogenous thiols are highly available). In
fact, in the absence of the vascular tissue, no significant
production of H2S was detected (Figure 3).
As the vasodilator effects of H2S are largely mediated by the
activation of KATP channels and consequent membrane
hyperpolarization of VSM cells,2−4 it was thought interesting
to evaluate the effects of 1 and NaHS on the membrane
potential of cultured human VSM cells (HASMCs). Fur-
thermore, the KATP-activator levcromakalim (LCRK) was
selected as reference hyperpolarizing agent. As shown in Figure
4, the administration of NaHS caused a significant membrane
hyperpolarization of HASMCs; the maximal hyperpolarization
Figure 5. Histograms represent the mean levels of systolic pressure,
recorded in normotensive rats, 20 min after the oral administration of
NaHS (0.1 mg/kg), compound 1 (equimolar dose), or their vehicle.
Data are calculated as % of the basal systolic blood pressure, recorded
before the administration. The vertical bars indicate the SEM; *** =
significantly different (P < 0.001) from vehicle.
In conclusion, this work furnished a small library of
thioamides showing different H2S-releasing rates. Some
derivatives (1−3 and 7) exhibited smart H2S-releasing
properties triggered by the presence of organic thiols, while
other derivatives (6 and 8−12) were thiol-independent H2S
donors. The p-hydroxybenzothioamide 1 produced typical
induced by 1 mM NaHS was 52
8% of that induced by
LCRK (10 μM). Compound 1 hyperpolarized the membranes
of VSM cells in a concentration-dependent fashion (Figures 3
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vascular effects of H2S, both in in vitro and in vivo experiments.
Specifically, compound 1, which was selected as representative
for investigating the potential vascular effects of all the
analogues of this series, inhibited the NA-induced vaso-
constriction in isolated rat aortic rings, hyperpolarized the
membranes of human HASMCs, and reduced the systolic
blood pressure after oral administration. These thioamide
derivatives might represent useful tools for the rational
development of promising H2S-releasing agents addressed to
the therapeutic treatment of cardiovascular diseases.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis, characterization, and pharmacological studies of
compounds 1−12. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: calderone@farm.unipi.it (V.C.); taliani@farm.unipi.it
(S.T.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This article has been supported by the ‘‘Regional Health
Research Program 2009’’ of Regione Toscana, Italy.
■
(18) Shukla, N.; Rossoni, G.; Hotston, M.; Sparatore, A.; Del
Soldato, P.; Tazzari, V.; Persad, R.; Angelini, G. D.; Jeremy, J. Y. Effect
of hydrogen sulphide-donating sildenafil (ACS6) on erectile function
and oxidative stress in rabbit isolated corpus cavernosum and in
hypertensive rats. BJU Int. 2009, 103, 1522−1529.
ABBREVIATIONS
■
DADS, diallyl disulfide; GYY4137, morpholin-4-ium 4-methox-
yphenyl-morpholino-phosphinodithioate; NA, noradrenaline;
SEM, standard error of mean; VSM, vascular smooth muscle
(19) Wallace, J. L.; Cirino, G.; Santagada, V.; Caliendo, G. Hydrogen
Sulfide Derivatives of Non-Steroidal Anti-Inflammatory Drugs.
WO2008/009127 A1, 2008.
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