Iminothioethers as Hydrogen Sulfide Donors: From the Gasotransmitter Release to the Vascular Effects

Article abstract of DOI:10.1021/acs.jmedchem.7b00888

The gasotransmitter hydrogen sulfide (H₂S) is an important tuner of the cardiovascular homeostasis, and its deficiency is etiologically associated with a number of cardiovascular diseases. Therefore, the research of original moieties able to release H₂S represents a timely issue for drug discovery. In this work, we developed a collection of iminothioethers (ITEs), exhibiting H₂S-releasing properties and producing vasorelaxing effects on rat aortic rings. Derivatives 4 and 11, selected as representative of slow and fast rate H₂S donors, respectively, produced a complete recovery of the basal coronary flow, reverting the AngII-induced effects in isolated rat hearts. In addition, studies on human aortic smooth muscle cells (HASMCs) demonstrated membrane hyperpolarizing effects, well related to the intracellular generation of H₂S. Taken together, the results obtained support ITEs 4 and 11 as new pharmacological tools, as well as effective and innovative H₂S donors for cardiovascular drug discovery.

Full text of DOI:10.1021/acs.jmedchem.7b00888

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Article
Iminothioethers as hydrogen sulfide donors: from
the gasotransmitter release to the vascular effects.
Elisabetta Barresi, Giulia Nesi, Valentina Citi, Eugenia Piragine, Ilaria Piano,
Sabrina Taliani, Federico Da Settimo, Simona Rapposelli, Lara Testai, Maria
Cristina Breschi, Claudia Gargini, Vincenzo Calderone, and Alma Martelli
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00888 • Publication Date (Web): 11 Aug 2017
Downloaded from http://pubs.acs.org on August 11, 2017
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Iminothioethers as hydrogen sulfide donors: from
the gasotransmitter release to the vascular effects.
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Elisabetta Barresi,†# Giulia Nesi,†# Valentina Citi,† Eugenia Piragine,† Ilaria Piano,† Sabrina
Taliani,† Federico Da Settimo,† Simona Rapposelli,† Lara Testai,† Maria Cristina Breschi,†
Claudia Gargini,† Vincenzo Calderone,† Alma Martelli,†*
†Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy.
# These authors equally contributed to the manuscript
* Corresponding author
KEYWORDS: hydrogen sulfide, vascular smooth muscle, iminothioethers, thioamides, H2Sꢀ
donors, vasorelaxation, gasotransmitter.
ABSTRACT: The gasotransmitter hydrogen sulfide (H2S) is an important tuner of the
cardiovascular homeostasis and its deficiency is etiologically associated with a number of
cardiovascular diseases. Therefore, the research of original moieties able to release H2S
represents a timely issue for drug discovery. In this work, we developed a collection of
iminothioethers (ITEs), exhibiting H2Sꢀreleasing properties and producing vasorelaxing effects
on rat aortic rings. Derivatives 4 and 11, selected as representative of slow and fast rate H2Sꢀ
donors respectively, produced a complete recovery of the basal coronary flow, reverting the
AngIIꢀinduced effects in isolated rat hearts. In addition, studies on human aortic smooth muscle
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cells (HASMCs) demonstrated membrane hyperpolarizing effects, well related with intracellular
generation of H2S. Taken together, the results obtained support ITEs 4 and 11 as new
pharmacological tools, as well as effective and innovative H2Sꢀdonors for cardiovascular drug
discovery.
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INTRODUCTION:
Hydrogen sulfide (H2S) is presently recognized as a fundamental mediator, which controls the
homeostasis of many biological systems in the mammalian body.1,2 This gasotransmitter is
biosynthetized by specific enzymes, such as cystathionineꢀbetaꢀsynthase (CBS), cystathionineꢀ
gammaꢀlyase (CSE) and 3ꢀmercaptopyruvate sulfurtransferase (3MST) starting from the
aminoacid LꢀCysteine. Among its numerous roles, H2S is a key regulator of the cardiovascular
system, where it is mainly produced by CSE. H2S acts as a vasodilator3 through several
mechanisms of action often involving the modulation of ion channels or phosphodiesterase
(PDE) in vascular smooth muscle.4ꢀ8 The deletion of CSE gene in experimental animals is
associated with a significant reduction of endogenous H2S in blood, and in vascular and
myocardial tissues; such a reduction leads to the impairment of endotheliumꢀmediated
vasorelaxation and increase in blood pressure.9 These data clearly indicate that vascular H2S is a
key factor in the regulation of blood pressure and the defective production of endogenous H2S is
likely to be one of the most important etiopathogenetic factor in several forms of hypertension.10
The roles played by endogenous H2S in the regulation of the cardiovascular homeostasis pave the
way to appealing therapeutic purposes, based on effective and rational pharmacological
modulation of the H2S pathway.11 Indeed, the administration of exogenous H2S has been proven
to exert significant antiꢀhypertensive effects in several experimental models of hypertension,12,13
indicating that "druggable" H2Sꢀreleasing agents can actually be viewed as promising tools to
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obtain novel cardiovascular drugs.14,15 The poor posological control and the high probability of
toxic effects strongly exclude the use of gaseous H2S. Some sulfide salts, such as sodium
hydrogen sulfide (NaHS) and calcium sulfide (CaS)16 are H2Sꢀgenerating agents widely used for
experimental purposes, but the rapid formation of H2S (due to the protonation of hydrosulfide
and sulfide anions, respectively, at physiological pH) seems to be poorly appropriate for clinical
uses. Ideal H2Sꢀdonor drugs should produce H2S with relatively slow and constant rates.
Accordingly, the search of novel H2Sꢀreleasing chemical moieties suitable for the development
of clinically effective H2Sꢀdonors is strongly required.
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An interesting H2Sꢀdonor feature has been early recognized in natural organosulfur derivatives,
such as the polysulfides of Alliaceae (for example, diallyl disulfide 1 (DADS), Chart 1).17 More
recently, H2Sꢀreleasing properties have been recognized also in another important class of
natural sulfur compounds: the isothiocyanates typical of Brassicaceae.18 Synthetic H2Sꢀreleasing
agents
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methoxyphenyl(morpholino)phosphinodithioatemorpholinium salt 2 (GYY4137, Chart 1) is one
of the most widely used in pharmacological studies.19 As well, H2Sꢀreleasing dithiolethiones and
thioamides (TAs) are largely used, especially for the synthesis of multitarget drugs.20ꢀ22
Satisfactory H2Sꢀreleasing features of aminothiol and aryl isothiocyanate derivatives have been
also reported.23,24
Very recently, original examples of “smart” H2Sꢀdonors, able to generate the gasotransmitter
based on specific mechanisms of release, which may be useful in specific biological targets, have
been described. Among these, molecules exhibiting esteraseꢀmediated production of H2S25, pHꢀ
controlled mechanisms26 or initial release of intermediates such as carbonyl sulfide27 have been
reported.
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In this context, we recently studied a small library of arylthioamides that exhibited satisfactory
properties, including stability in water and relatively slow H2S generation, triggered by the
presence of organic thiols.28 Slight structural modifications, such as the insertion of small
substituents in the benzene ring or the replacement of the benzene ring with heterocycles,
afforded different rates of H2S release, even comparable to or even higher than that of 2.28 In
addition, a compound from this series produced typical vascular effects of H2S, both in in vitro
and in vivo experiments, including: (i) inhibition of the norepinephrineꢀinduced vasoconstriction
in isolated rat aortic rings; (ii) membrane hyperpolarization in human vascular smooth muscle
cells; (iii) reduction of the systolic blood pressure after oral administration.28
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Despite the huge amount of data on the potential pharmacological usefulness of H2S donors and
H2Sꢀhybrids, to date, there is poor heterogeneity of H2Sꢀreleasing moieties. Thus, the
development of original H2Sꢀdonors characterized by varying physicochemical, biological and
pharmacological features represents a very timely issue for drug discovery.
In this paper, we report the synthesis and the pharmacological evaluation of some iminothioether
derivatives (ITEs 3-11, Chart 1), with the aim to investigate such a novel and original chemical
moiety as a H2Sꢀreleasing functional group with potential pharmaceutical interest. In addition, a
small collection of closely analogous TAs (12-15, Chart 1) were synthetized and investigated.
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Chart 1. Chemical structures of reference H2Sꢀdonors (1, 2) and of the novel synthetized
compounds 3-15.
CHEMISTRY
As reported in Scheme 1A, the synthetic procedure for the preparation of the target
benzimidothioate derivatives 3-5 started from the commercially available benzamide 18 or 4ꢀ
methoxybenzamide 19, obtained through a condensation between the appropriate benzoyl
chloride (16 or 17) and NH4OH in the presence of triethylamine. Compounds 18 and 19 were
allowed to react with Lawesson’s reagent in dry THF solution, to give products 20 and 21
. Compounds 3 and 4 were then obtained by alkylation of compounds 20 and 21, respectively,
with benzylbromide in refluxing CHCl3. Finally, compound 4 was demethylated by treatment
with BBr3 in nitrogen atmosphere, to obtain compound 5 (Scheme 1A).
The preparation of the target Nꢀbenzylbenzothioamide derivatives 12-14 started from the
commercially available Nꢀbenzylbenzamide 22, or Nꢀbenzylꢀ4ꢀmethoxybenzamide 23 obtained
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through a condensation between 4ꢀmethoxybenzoyl chloride and benzylamine in the presence of
triethylamine. Compounds 22 and 23 were allowed to react with Lawesson’s reagent in dry THF
solution to give products 12 and 13, respectively. Compound 13 was finally demethylated by
treatment with BBr3, under nitrogen atmosphere, to obtain compound 14 (Scheme 1A).
The arylimidothioate derivatives 6-8 were prepared by treatment of the appropriate 4ꢀsubstituted
benzonitrile 24-26 with thiophenol under an atmosphere of HBr, as previously reported by Baati
et al.29(Scheme 1B).
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Scheme 1. Synthesis of imithioether 3-8 and thioamide 12-14 derivatives.
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Reagents and conditions. I: NH4OH, NEt3, dry toluene, 24 h, r.t.; II: Lawesson’s reagent, dry
THF, 12 h, r.t.; III: benzylbromide, NaH, CHCl3, 12 h, reflux; IV: BBr3, dry CH2Cl2, 24 h, r.t.;
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V: benzylamine, NEt3, dry toluene, 24 h, r.t.; VI: Lawesson’s reagent, dry THF, 12 h, r.t.; VII:
BBr3,dry CH2Cl2, 24 h, r.t.; VIII: thiophenol, Et2O, HBr, 0 °C, 0.5 h.
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The experimental procedures for the preparation of compounds 9-11, 15 are outlined in Scheme
2.
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The thiophene derivatives 9, 10 and 15 were synthesized as reported in Scheme 2A. Reaction of
2ꢀthiophenecarboxamide 27 with the Lawesson’s reagent in chlorobenzene yielded the
corresponding thioamide 28, which was then condensed with benzylbromide or naphthꢀ2ꢀ
ylmethylbromide to give the target products 9 and 10, respectively. The thioamide 15 was
obtained by two sequential reactions of 2ꢀthiophenecarboxamide 27 with benzyl bromide, in the
presence of NaH, and then with Lawesson’s reagent (Scheme 2A).
The phenyl thiopheneꢀ2ꢀcarbimidothioate 11 was prepared starting from the 2ꢀthiopheneꢀnitrile
30 by the same procedure applied for compounds 6-8.29(Scheme 2B).
All the target compounds 3-15 were finally purified by flash chromatography, when necessary
(see Experimental section).
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Scheme 2. Synthesis of imithioether 9-11 and thioamide 15 derivatives.
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Reagents and conditions: I: Lawesson’s reagent, C6H5Cl, 12 h, 130 °C; II: appropriate
arylbromide, CHCl3, 12 h, reflux; III: benzyl bromide, NaH, DMF, 1 h, r.t.; IV: Lawesson’s
reagent, C6H5Cl, 12 h, 130 °C; V: thiophenol, Et2O, HBr, 0 °C, 0.5 h.
RESULTS AND DISCUSSION
Evaluation of H2S-release by the amperometric assay.
The investigation of the H2Sꢀreleasing properties of the novel synthesized compounds was
carried out in vitro by an amperometric assay, by means of a H2Sꢀselective minielectrode, to
have a realꢀtime determination of the H2Sꢀrelease and thus to perform a qualitative/quantitative
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description of the process. Table 1 lists the parameters of Cmax (the highest concentration
achieved in the recording time) and t1/2 (the time required to reach a concentration = ½ Cmax)
from the tested compounds (incubated at the concentration 1mM), recorded in the absence (ꢀLꢀ
Cys) or in the presence (+LꢀCys) of an excess of LꢀCysteine (4 mM). Data of reference H2Sꢀ
donors 1 and 2 were also reported for comparison purposes.
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In general, all the compounds (ITEs and TAs) showed very poor H2Sꢀrelease in the absence of Lꢀ
Cys, consistently with the H2Sꢀreleasing profile exhibited by the reference H2Sꢀdonors (1 and 2),
in previous experiments.28 In particular, in the absence of LꢀCys, the H2Sꢀgeneration from the
ITE 10 and TA 13 was almost negligible (under the levels of determination), while all the other
compounds and the reference H2Sꢀdonors exhibited low but evident release of H2S, although in
some cases it was under the level of accurate quantification (compounds 11, 14, 15, 1 and 2).
The preꢀincubation with an excess of LꢀCys (4 mM) improved the H2Sꢀrelease from almost all
the synthesized molecules, as well as from 1 and 2. In particular, the maximal concentrations of
H2S (Cmax), generated from the tested compounds upon incubation (for 30 min) in the presence
of LꢀCys, ranked from 0.31 µM (15) to 19.0 µM (11); the Cmax of 1 was 19.4 µM. In the
presence of LꢀCys, H2Sꢀrelease from compound 13 was low but evident (under the level of
accurate quantification), while no detectable release of H2S was recorded for 10 (Table 1).
Looking at the LꢀCys mediated effects, almost all the compounds showed progressive and timeꢀ
related "slow" H2Sꢀreleasing profiles, with t1/2 values ranging from 4.4 and 11.9 min. The H2Sꢀ
releasing profile of 4 is shown in Figure 1, as representative example; in previously published
data, 1 exhibited a relatively faster, but clearly timeꢀrelated, H2Sꢀreleasing process, with t1/2
value of 1.5 min.28 Compound 11 exhibited a profile of relatively "rapid" donor (t1/2 = 0.28 min,
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Table 1), reaching the peak concentration (19.0 µM) in about 1 min, followed by a progressive
decrease of the H2S concentration (Figure 1).
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Taken together, these data indicate that, in the presence of organic thiols (LꢀCys), most of the TA
and ITE compounds behaved as H2Sꢀdonors, albeit with different features both in the
quantitative and in the kinetic aspects that can be related to their chemical structure. Specifically,
the “rank order” for quantitative H2S release within the ITEs series was: 11 >4 ≥5 >9 >3 >10.
From a structureꢀactivity relationship point of view, it could be observed that the insertion of a
substituent (OCH3, OH) on the phenyl ring featuring the iminothioether function of 3 determines
a slight improvement in the H2Sꢀreleasing properties in compounds 4 and 5. The replacement of
the same phenyl ring of 3 with a thienyl moiety in 9 did not produce significant effects on Cmax
values. On the contrary, the direct comparison between 11 and 9 indicates that the Sꢀphenyl
substitution is highly effective in increasing the quantitative H2S release and its rate; in addition,
this effect seems to be due to the presence of the phenyl substituent on the S atom, rather than the
thiophene ring, as compound 9 exhibited almost equivalent levels of H2S release if compared
with phenyl derivatives 3-5. Replacement of the benzyl group in compound 9 with a naphthꢀ2ꢀ
ylmethyl moiety gives compound 10 that is devoid of any H2Sꢀreleasing activity, probably due to
chemicalꢀphysical issues connected to its high lipophilicity.
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Finally, the H2S release from TA compounds was significant, but quite low (Table 1). The
comparison between the ITEs 3-5, 9 and the corresponding analogous 12-15, indicates that the
former functional group leads to a general quantitative improvement in H2S release, suggesting
ITE as a novel original H2Sꢀdonor group useful in the future development of innovative
cardiovascular drugs.
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Noteworthy, it is widely accepted that the biological activity of H2Sꢀdonors is not directly related
with the mere quantitative aspects of the release: indeed, even small amounts of H2S generated in
a longꢀlasting manner, can evoke effects better than those evoked by fast and transient generation
of large amounts of the gasotransmitter. This has been described for slow H2Sꢀdonors, such as
219 or some arylꢀisothiocyanates,24 which caused vasorelaxing effects with potency values higher
than sodium hydrosulfide, a widely used salt that instantaneously generates H2S at physiological
pH. As well, the effects of H2S donors on inflammatory processes are strongly influenced by the
rate of the H2S release, and again slow H2Sꢀdonors exhibited more favorable profiles of
activity.19
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Therefore, ideal H2S donors for many clinical uses should produce H2S with slow releasing rates,
ensuring low and longꢀlasting concentration.14 In this perspective, the LꢀCysꢀinduced H2Sꢀ
release from almost all the compounds tested in this study exhibited quite "slow" rate,
comparable and even longer than those exhibited by wellꢀknown donors, such as 1 and 2 (see
Table 1), when tested in the same experimental conditions.28 Only compound 11 exhibited a
short t1/2 value (< 1 min), generating an early peak of H2S concentration, followed by a
progressive decrease (Figure 1).
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Comp. 4 100µM +L-Cys 4mM
Comp. 11 100µM +L-Cys 4mM
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Figure 1. Amperometric recordings of the H2Sꢀrelease from 4 and 11 in the presence of LꢀCys
(+ LꢀCys). The curves describe the increase of the H2S concentration with respect to time,
following the incubation of tested compounds, highlighting a slow and gradual increase of the
H2S production after 4 administration and, conversely, a rapid and massive H2S production
followed by a rapid decrease after 11 administration; the vertical bars indicate SEM. Twoꢀway
ANOVA showed extremely significant difference between the two curves (P < 0.001).
Functional evaluation of the vasorelaxant effects on rat aortic rings.
The vasorelaxing effects of ITEs and TAs were tested on preꢀcontracted rat aortic rings. Almost
all the tested compounds showed full or almost full vasorelaxant efficacy (Emax) on
endotheliumꢀdenuded rat aortic rings preꢀcontracted with 25 mM KCl, with potency indexes
(pIC50) ranging between 3.20 and 3.89 (Table 1). The vasorelaxing effects were also exhibited by
compounds 13. This compound released well detectable, but very low (under the level of
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accurate quantification), H2S concentrations in the amperometric assay. Therefore, it cannot be
excluded that the vasorelaxing activity can be attributable even to other mechanisms different
from H2S release. Consistent with the data previously obtained, compound 10, which lacks of
any detectable H2Sꢀreleasing activity, showed very poor vasorelaxant activity and a nonꢀ
calculable potency index, strongly suggesting that the vasorelaxing effect of the other
compounds is actually mediated by H2S.
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N
H2S-release
Vasorelaxing Effect
ꢀLꢀCys
+LꢀCys 4mM
pIC50
Emax
t1/2(min)
t1/2(min)
Cmax (µM)
Cmax (µM)
< 0.30
N.C.
N.C.
19.4 ± 5.5
1.5 ± 0.3
2.5 ± 0.8
5.23 ± 0.58
5.73 ± 0.52
5.10 ± 0.62
ꢀ
41.7 ± 1.7
60.2 ± 3.7
86.5 ± 3.6
89.9 ± 3.3
82.1 ± 3.4
N.T. a
N.C.
1
2
< 0.30
10.3 ± 2.6
3.20 ± 0.50
7.20 ± 0.80
6.80 ± 0.80
N.T. a
3.68 ± 0.01
3.37 ± 0.02
3.59 ± 0.02
3.31 ± 0.01
ꢀ
2.30 ± 0.40
1.18 ± 0.30
0.50 ± 0.10
N.T.a
3.61 ± 0.38
17.35 ± 2.90
5.28 ± 0.71
ꢀ
3
4
5
6
N.T. a
ꢀ
N.T. a
ꢀ
N.T.a
ꢀ
7
N.T. a
ꢀ
N.T.a
ꢀ
N.T. a
ꢀ
8
1.90 ± 0.1
N.D.
26.91 ± 4.18
N.D.
4.60 ± 0.90
N.D.
6.66 ± 0.73
N.D.
89.9 ± 2.1
20.4 ± 3.3
99.7 ± 0.3
92.7 ± 0.4
96.8 ± 0.6
88.2 ± 0.4
91.7 ± 0.7
3.58 ± 0.03
N.C.
9
10
11
12
13
14
15
< 0.30
10.24 ± 2.11
8.70 ± 1.20
N.D.
19.0 ± 4.90
1.90 ± 0.40
< 0.30
0.28 ± 0.13
11.86 ± 1.35
N.C.
3.20 ± 0.01
3.79 ± 0.03
3.89 ± 0.01
3.46 ± 0.02
3.62 ± 0.03
0.70 ± 0.10
N.D.
< 0.30
1.25 ± 0.23
4.3 ± 0.71
1.10 ± 0.10
0.31 ± 0.09
4.43 ± 0.30
7.53 ± 0.70
< 0.30
Table 1. Parameters of Cmax and t1/2, emerging from the amperometric detection of H2Sꢀrelease
from the tested compounds (incubated at the concentration 1mM) in the absence (ꢀLꢀCys) or in
the presence (+LꢀCys) of an excess of LꢀCys (4 mM), and parameters of Emax (maximal
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vasorelaxing effect evoked by the tested compounds 1mM) and pIC50 of the vasorelaxing effects
recorded on preꢀcontracted rat aortic rings. In the amperometric detection, the lower limit of
reliable quantitative determination of H2S was 0.3 ꢁM. Data are expressed as means ± standard
error. N.T.= Not tested (chemically unstable in experimental conditions); N.D. = not detectable;
N.C. = not calculable. All the synthetized compounds, except compound 10, exhibited
vasorelaxing efficacy parameters significantly higher (P<0.01) than 1 and 2.
10
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Functional evaluation of the effects on coronary flow.
On the basis of the results emerging from the amperometric assay and the functional data on the
aortic rings, two iminothioether derivatives, 4 and 11, were selected as representative for further
pharmacological investigation on the basis of the following issues: (i) 4 exhibited high
vasorelaxant potency, appreciable quantitative H2Sꢀrelease and a slow releasing rate; (ii) 11
showed lower vasorelaxant potency, but it generated the highest concentration of H2S, with a
quite fast rate. The two selected compounds were then evaluated in Langendorffꢀperfused rat
hearts. As expected, the perfusion with Angiotensin II (AngII, 0.1 µM) caused a significant
reduction (by about 25%) of the coronary flow (CF) in isolated rat hearts when compared to the
basal CF (basal flow = 10.45 ± 0.64 ml/min/g). The "addꢀon" perfusion with the ITE 4 (300 µM)
produced extremely significant effects in the coronary bed, leading to an intense increase of the
coronary flow, up to 165%, i.e. higher than the basal one. (Figure 2). In contrast, perfusion with
11 (300
µM) led to an apparent increase of the coronary flow, but this effect was not
significantly different than that induced by the vehicle (Figure 2).
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AngII
TC
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Vehicle
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Comp. 4 300 µM
Comp. 11 300 µM
***
NS
0
5
10 15 20 25 30 35 40
Time (min)
Figure 2. Changes (in %) of CF, induced by perfusion of AngII, followed by the addꢀon
perfusion of vehicle, 4 or 11. After the equilibration time, three measurements of the basal flow
were carried out at 5 min intervals, starting from min 0. Immediately after the recording of the
third basal value, AngII was perfused from min 10; the perfusion with AngII was maintained
until the end of the experiment (upper bar). Starting from min 25, the tested compound (TC) or
the vehicle were perfused (lower bar), together with AngII. Data are expressed as a % of the
mean basal coronary flow, and are expressed as means± standard error, from hearts of 6ꢀ9
animals. NS = the differences between the curves are not statistically significant; *** the
differences between the curves are extremely significant (P < 0.001).
Evaluation of membrane hyperpolarization of human aortic smooth muscle
cells (HASMCs).
Among the heterogeneous mechanisms of action accounting for the vasorelaxant activity of H2S,
the activation of ATPꢀsensitive potassium (KATP) channels5,30 and of vascular Kv7 potassium
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channels,4 with membrane hyperpolarization of vascular smooth muscle cells, seem to play a
relevant role. In this view, it was thought interesting to evaluate the effects of 4 and 11 on the
membrane potential of cultured human vascular smooth muscle cells (HASMCs), taking 1,3ꢀ
dihydroꢀ1ꢀ[2ꢀhydroxyꢀ5ꢀ(trifluoromethyl)phenyl]ꢀ5ꢀ(trifluoromethyl)ꢀ2Hꢀbenzimidazolꢀ2ꢀone 31
(NS1619), a wellꢀknown potassium channel activator, as reference hyperpolarizing agent31.
3
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Both ITEs 4 and 11 (100 µM ꢀ 1 mM) caused a significant and concentrationꢀrelated membrane
hyperpolarization of HASMCs. In particular, the hyperpolarizing response evoked by the highest
tested concentration of 11 (1mM) was significantly lower (36 ± 5 %) than that evoked by the
reference 31 (Figure 3). Actually, in previous works, we could observe that even the fast H2Sꢀ
donor, sodium hydrosulfide, evokes moderate hyperpolarizing effects.4,24 Compound 4 evoked
strong membrane hyperpolarization, exhibiting high level of efficacy (149 ± 2%) and
significantly overcoming 31 (Figure 3).
The effects observed on the coronary flow, in rat isolated hearts, and on the membrane potential
of HASMCs witness again that a "slow" and moderate H2Sꢀrelease seems to be preferable to a
“fast” H2Sꢀrelease for the vascular effects.
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*
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*
50
*
*
*
*
*
*
*
*
*
*
25
0
Figure 3. The graph shows the hyperpolarizing effect of tested compounds on sarcolemmal
membrane of HASMCs. Data are expressed as mean ± standard error. Six different experiments
were performed, each in six replicates. Asterisks indicate significant difference from the effect
evoked by 31 (* = P < 0.05; ** = P < 0.01; *** = P < 0.001).
Evaluation of H2S-release in HASMCs.
The amperometric technique well defines the kinetics (i.e., the rate) of the LꢀCysꢀdependent H2Sꢀ
release of compounds 3-5 , 9-15, suggesting that many of these may act as "smart" donors: they
are expected to be relatively stable in water, but they behave as H2Sꢀgenerating agents in
biological environments (for example, the cell cytosol), where they can interact with endogenous
organic thiols (LꢀCys, glutathione, etc). However, this assay was carried out only in buffer
aqueous solution in the absence and in the presence of LꢀCys. Therefore, a further evaluation
was performed in order to demonstrate that the H2Sꢀrelease actually occurs in cells, without
adding exogenous thiols. In particular, the H2S generation was detected in HASMCs by
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spectrofluorometric measurements using the dye 3'ꢀmethoxyꢀ3ꢀoxoꢀ3Hꢀspiro[isobenzofuranꢀ1,9'ꢀ
xanthen]ꢀ6'ꢀyl 2ꢀ(pyridinꢀ2ꢀyldisulfanyl)benzoate (Washington State Probeꢀ1,WSPꢀ1), which
specifically and irreversibly interacts with H2S.32 The fluorescence produced by this interaction
was quantitatively recorded by a spectrofluorometric approach, and also observed by
fluorescence microscopy.
10
11
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Spectrofluorometric measurements showed that the addition of the vehicle did not cause any
significant increase of fluorescence. In contrast, the addition of 1mM 11 to HASMCs, preꢀ
loaded with the fluorescent dye WSP1, led to a massive timeꢀdependent increase of fluorescence
(FI, fluorescence index), indicating an extremely significant generation of H2S (P<0.01 vs
vehicle). The maximal value of FI, recorded after 1 h of incubation, was about 50ꢀfold higher
than that evoked by 300 µM reference 1 (Figure 4A). After 1 h of incubation, the fluorescence
increase reached an apparently stable "steady state", suggesting that the H2Sꢀreleasing process is
completed, and thus confirming the profile of "fast" donor for 11.
In contrast, the addition of 1mM 4 to WSP1ꢀpreloaded HASMCs led to a significant (P<0.01 vs
vehicle) but moderate timeꢀdependent increase of FI. The maximal level of FI recorded after 1 h
of incubation was significantly lower (P < 0.01) than that evoked by 11 (1 mM) and was almost
completely comparable to that evoked by 1 (300 µM). In addition, after 1 h of incubation, the
fluorescence increase did not yet reach a "steady state" and was still in progress, indicating that
the H2Sꢀreleasing process is not complete, thus confirming the profile of "slow" donor for 4
(Figure 4B).
Fluorescence microscopy allowed us to observe a clear increase of WSPꢀ1ꢀevoked fluorescence
inside the HASMCs treated with 4, indicating an intracellular localization of the H2S release
from this ITE (Figure 5). 1 showed a similar feature (Figure 5). In contrast, a significant cell loss
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was observed in HASMCs treated with 11 (data not shown), suggesting that ITE may have
caused cell damage and consequent vulnerability in the experimental procedures used in the
microscopy approach, which are more "invasive" if compared with the spectrofluorometric
technique.
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A
250000
Vehicle
Comp. 11 1mM
Comp. 1 300µM
200000
150000
100000
50000
0
***
0
10
20
30
40
50
60
min
B
8000
6000
4000
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0
Vehicle
Comp. 4 1mM
Comp. 1 300µM
***
***
0
10
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Figure 4. The graphs show the WSPꢀ1 fluorescence increase evoked by the administration of
vehicle, 11 and 1 (A), 4 and 1 (B) on HASMCs. Data were expressed as mean ±standard error.
Three different experiments were carried out, each in triplicate. *** = significantly different
from the vehicle (P < 0.01).
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-
Figure 5. Fluorescence microscopy images, showing the fluorescence evoked in HASMCs (preꢀ
loaded with WSPꢀ1 dye), after the administration of vehicle, compound 4 (300ꢁM) and 1 (300
ꢁM). The green fluorescence indicates a significant activation of the fluorophore, due to the
generation of H2S and its interaction with WSPꢀ1. Propidium iodide was used to identify the
nuclei, in red.
Evaluation of cGMP in HASMCs.
Intracellular contents of cGMP in HASMCs were determined by ELISA assay. In basal
conditions no detectable levels of cGMP could be observed both in vehicle treated cells and also
in HASMCs treated with the selected H2Sꢀdonors. In contrast, in sodium nitroprusside (SNP)
preꢀtreated cells treated with the vehicle (DMSO 0.1%) wellꢀdetectable and significant
concentration of cGMP was detected (0.446 ± 0.005 pmol/ml). The administration of compound
4 to SNPꢀpretreated cells, led to small but significant increase in cGMP concentration (0.489 ±
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0.014 pmol/ml). Finally, compound 11 determined in SNP_pretreated cells a larger and
5
6
significant increase of cGMP intracellular concentration (0.667 ± 0.021 pmol/ml).
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0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
***
*
Figure 6. Effects of selected H2Sꢀdonors on intracellular increase of cGMP. HASMC were preꢀ
treated with SNP (1mM) and then incubated with vehicle (DMSO 0.1%), compound 4 or
compound 11 (300 ꢁM), for 20 min. Data are expressed as mean ± SEM. The asterisks indicate
significant differences vs vehicle (* = P < 0.05; *** = P < 0.001).
Effects of selected H2S-donors on blood pressure.
Normotensive Wistar rats showed basal systolic pressure (Psys) of 137 ± 2 mmHg.
Intraperitoneal administration of LꢀNAME (100 mg/Kg) caused a significant increase of Psys
158 ± 2 mmHg. The administration of vehicle (DMSO 0.33ml/Kg i.p.) did not cause any
significant change of Psys in rats with LꢀNAMEꢀinduced hypertension. In contrast, the reference
H2Sꢀdonor 1 (133 µmol/Kg) caused a significant (P < 0.01) decrease of Psys (ꢀ39 ± 5%).
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Compound 4 (133 µmol/Kg) exhibited a similar pharmacological behavior. In particular, it
promoted a significant (P < 0.01) decrease of Psys (ꢀ36 ± 4%) in LꢀNAMEꢀinduced hypertensive
animals. Compound 11 (133 µmol/Kg) did not influence the Psys.
10
11
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0
-10
-20
-30
-40
**
-50
**
Figure 7. Changes in Psys (expressed as a % of the LꢀNAMEꢀinduced hypertensive Psys),
following the i.p. administration of compounds 4 and 11, the reference H2Sꢀdonor 1 or the
corresponding vehicle. The asterisk indicates significant difference vs vehicle (** = P < 0.01).
CONCLUSIONS
In this work, the ITE group was investigated as potential new H2Sꢀreleasing moiety, in view of
its similarity with TA (a consolidated H2Sꢀdonor moiety). Thus, a small collection of ITE
derivatives were synthesized and pharmacologically characterized.
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In the amperometric assay, many of the ITE derivatives behaved as smart H2Sꢀdonors (quite
stable in water, but able to generate H2S in the presence of LꢀCys, mimicking the endogenous
thiols), and showed a general increase in H2S release with respect to their TA analogues. Thus,
the H2Sꢀdonor profile of ITEs can be considered as an original and innovative finding of this
work. In addition, almost all the TAs and ITEs exhibited full vasorelaxing effects when tested on
preꢀcontracted rat aortic rings, with pIC50 values that significantly correlate with t1/2 values.
On the basis of the H2Sꢀreleasing profile emerging from the amperometric assay (i.e., a “cellꢀ
free” experimental model), two iminothioether derivatives, 4 and 11, were selected for further
pharmacological investigation, as representative of slow and fast rate H2Sꢀdonors, respectively.
To demonstrate the ability of ITEs to release H2S in a cellꢀbased experimental model, without the
adding of exogenous thiols, the H2S generation from the compounds in HASMCs was
investigated by means of a spettrofluorometric approach. The incubation of the two derivatives
on WSPꢀ1ꢀpreloaded HASMCs confirmed the profile of "fast" and "slow" donor, respectively for
4 and 11. In particular, the incubation of 11 led to a massive increase of FI, indicating a
dramatically efficient production of H2S inside cells. In contrast, compound 4 led to a moderate
and more gradual increase of fluorescence, related with the intracellular production of H2S.
Fluorescence microscopy confirmed the intracellular localization of the H2Sꢀgeneration for 11.
As concerns the functional pharmacological effects, the "addꢀon" perfusion in isolated hearts
with 11 showed an apparent trend to increase the coronary flow, however, this effect did not
reach the level of statistical significance. In contrast, in the same experiments, 4 exhibited
stronger effects, evoking an intense and extremely significant increase of coronary flow, up to
165%, i.e. higher than the basal one.
10
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The possible involvement of membrane hyperpolarizing effects in the H2Sꢀinduced
vasorelaxation by 4 and 11 was also investigated. The results confirmed that the slow H2S
releasing compound 4 is able to promote strong and concentrationꢀdependent hyperpolarizing
responses, exhibiting high level of efficacy and significantly overcoming the reference
hyperpolarizing drug 31. While, the fast H2Sꢀdonor 11 showed lower hyperpolarizing effects.
Beside the membrane hyperpolarizing effects, even the inhibition of phosphodiesterases (PDEs)
and consequent rise of intracellular cGMP are recognized as relevant mechanisms of action
accounting for the vasorelaxing effects of H2S6,33. Thus, the effects of compounds 4 and 11 on
the intracellular levels of cGMP have been also investigated. In HASMC, the guanylate cyclase
activation was triggered by the nitric oxide donor SNP and the concentration of cGMP was
significantly increased by the tested compounds, suggesting a probable involvement of H2Sꢀ
mediated inhibition of PDE. In particular, compound 11 evoked the higher effect seeming to
indicate that a more rapid release would be preferable for this specific effects and further
stressing that both the different kinetics of release and the H2S concentration may influence the
overall pharmacodynamic profiles of H2Sꢀdonors.
10
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Finally, the in vivo antiꢀhypertensive effects of 4 and 11 were evaluated in an experimental
model of LꢀNAMEꢀinduced hypertension. In this experimental protocol, the slow H2Sꢀdonor 4
evoked marked hypotensive activity, fully comparable with those shown by the reference drug 1.
In contrast, the fast donor 11 did not promote any significant decrease of blood pressure.
Taken together, the data reported in this work demonstrated that ITE can be considered as a
satisfactory new H2Sꢀreleasing moiety and that ITEꢀbased compounds, endowed with
appropriate H2Sꢀreleasing profile, may represent an original and promising class of “smart” H2Sꢀ
donors for the development of cardiovascular drugs.
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EXPERIMENTAL SECTION
5
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Chemistry
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General Material and Methods. Melting points were determined on a Kofler hotꢀstage apparatus
and are uncorrected. Chemical shifts (δ) are reported in parts per million downfield from
tetramethylsilane and referenced from solvent references; coupling constants J are reported in
hertz. 1H NMR and 13C NMR spectra of all compounds were obtained with a Varian Gemini 200
10
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MHz or a Bruker TopSpin 3.2 400 MHz spectrometer. C NMR spectra were fully decoupled.
The following abbreviations are used: singlet (s), doublet (d), triplet (t), double−doublet (dd),
and multiplet (m). Chromatographic separation was performed on silica gel columns by flash
(Kieselgel 40, 0.040−0.063 mm; Merck) or gravity column (Kieselgel 60, 0.063−0.200 mm;
Merck) chromatography. The ≥95% purity of the tested compounds was determined by HPLC,
using a Shimadzu LCꢀ20AD SP liquid chromatograph equipped with a DDA Detector at 196 nm
(column C18 (250 mm x 4.6 mm, 5 µm, Shimꢀpack)); the mobile phase, delivered at isocratic
flow, consisted of methanol (70–80%) and water (20–30%) and a flow rate of 1.0 mL/min.
Reactions were followed by thinꢀlayer chromatography (TLC) on Merck aluminum silica gel (60
F254) sheets that were visualized under a UV lamp. The microwaveꢀassisted procedures were
carried out with a CEM Discover LabMate microwave. Evaporation was performed in vacuo
(rotating evaporator). Sodium sulfate was always used as the drying agent. Commercially
available chemicals were purchased from SigmaꢀAldrich.
Benzyl benzimidothioate (3). A solution of benzylbromide (0.17 mL, 1.40 mmol) in 1.0 mL of
CHCl3 was added dropwise to a stirred solution of benzothioamide 20 (0.200 g, 1.40 mmol) in
3.0 mL of the same solvent. The reaction mixture was heated to reflux for 12 h (TLC analysis).
Then the solvent was concentrated under reduced pressure. Then diethylether was added until the
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formation of a precipitate, which was collected by vacuum filtration and then washed with cold
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ether. Yield: 55%; mp = 168ꢀ170 °C; H NMR (DMSOꢀd6, ppm): 4.75 (s, 2H); 7.36ꢀ7.46 (m,
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3H); 7.52ꢀ7.54 (m, 2H); 7.63ꢀ7.67 (m, 2H); 7.79ꢀ7.83 (m, 1H); 7.89ꢀ7.91 (m, 2H). 13C NMR
(DMSOꢀd6, ppm): 37.34; 128.87; 128.95; 129.53; 129.92; 130.01; 131.44; 133.38; 135.75;
186.98.34
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Benzyl 4-methoxybenzimidothioate (4). Compound 4 was obtained from compound 21 (0.234
g, 1.40 mmol) and benzylbromide (0.17 mL, 1.40 mmol) following the same procedure described
for 3 . The crude product was collected by vacuum filtration and then washed with cold ether.
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Yield: 52 %; mp = 195ꢀ197 °C; H NMR (DMSOꢀd6, ppm): 3.89 (s, 3H); 4.70 (s, 2H); 7.18 (d,
2H, J = 8.8 Hz); 7.36ꢀ7.45 (m, 3H); 7.51ꢀ7.54 (m, 2H); 7.95 (d, 2H, J = 8.8Hz); 11.69 (bs, exch.
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D2O, 1H). C NMR (DMSOꢀd6, ppm): 37.14; 56.59; 115.55; 122.80; 128.93; 129.52; 129.92;
131.62; 133.45; 165.91; 185.46.34
Benzyl 4-hydroxybenzimidothioate (5). To
a
stirred suspension of benzylꢀ4ꢀ
methoxybenzimidothioate 4 (0.270 g, 1.00 mmol) in 10.0 mL of dry dichloromethane, cooled at
ꢀ10 °C, a solution of BBr3 (1.26 mL, 7.31 mmol) in 1.0 mL of the same solvent, was added
dropwise. The mixture was left under stirring at room temperature for 24 h under nitrogen
atmosphere (TLC analysis). The solvent was evaporated at reduced pressure, and the solid
precipitate was washed several times with methanol. The product was finally purified by flash
chromatography eluting with petroleum ether 60ꢀ80 °C/AcOEt (7/3). Yield: 60%; mp = 133ꢀ136
°C; 1H NMR (DMSOꢀd6, ppm): 4.32 (s, 2H); 6.82 (d, 2H, J= 8.2 Hz); 7.28ꢀ7.44 (m, 5H); 7.65 (d,
2H, J= 8.4 Hz); 11.09 (bs, exch. D2O, 1H); 11.52 (bs, exch. D2O, 1H). 13C NMR (DMSOꢀd6,
ppm): 33.96; 115.78; 127.69; 128.01; 128.98; 129.30; 129.42; 129.90; 160.58; 168.09.
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Phenyl benzimidothioate hydrobromide (6). A solution of benzonitrile 24 (0.94 g, 9.16 mmol)
and thiophenol (0.93 ml, 9.16 mmol) in Et2O (1mL) was kept under an atmosphere of HBr in an
iceꢀbath. A precipitate separated very quickly. After stirring for 0.5 h at r.t., the white precipitate
was filtered and washed with Et2O. The crude compound was sufficiently pure to be used
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without further purification. Yield: 41%; white hygroscopic solid. H NMR (DMSOꢀd6, ppm):
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7.71ꢀ7.62 (m, 5H); 7.78ꢀ7.83 (m, 3H); 7.95ꢀ7.97 (m, 2H). C NMR (DMSOꢀd6, ppm): 168.33,
136.23, 134.72, 131.66, 129.95, 128.66, 128.06, 127.91, 127.66.
4-Methoxyphenyl benzimidothioate hydrobromide (7). Compound 7 was obtained from 4ꢀ
methoxybenzonitrile 25 (1.22 g, 9.16 mmol) and thiophenol (0.93 mL, 9.16 mmol) in Et2O
1
(1mL) following the same procedure described for 6. Yield: 55%; mp = 198ꢀ200 °C. H NMR
(DMSOꢀd6, ppm): 3.85 (s, 3H); 7.21 (d, 2H, J = 9.0 Hz); 7.63ꢀ7.70 (m, 3H); 7.76ꢀ7.78 (m, 2H);
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8.00 (d, 2H, J = 9.0 Hz). C NMR (DMSOꢀd6, ppm): 165.78; 135.98; 132.68; 131.78; 131.43;
123.73; 122.56; 115.42; 56.59.
4-Hydroxyphenyl benzimidothioatehydrobromide (8). Compound 8 was obtained from 4ꢀ
hydroxybenzonitrile 26 (1.09 g, 9.16 mmol) and thiophenol (0.93 mL, 9.16 mmol) in Et2O (1mL)
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following the same procedure described for 6.Yield: 65%; mp = 204ꢀ206 °C. H NMR (DMSOꢀ
d6, ppm): 7.01 (d, 2H, J = 7.8 Hz); 7.63ꢀ 7.72 (m, 3H); 7.76ꢀ 7.78 (m, 2H); 7.93 (d, 2H, J = 7.8
Hz); 11.09 (bs, exch. D2O, 1H). 13C NMR (DMSOꢀd6, ppm): 165.46; 135.98; 132.64; 132.21;
131.42; 123.69; 120.58; 116.81.
Benzyl thiophene-2-carbimidothioate (9). Benzyl bromide (0.360 g, 2.10 mmol) was added to
a solution of the compound 28 (0.300 g, 2.10 mmol) in CHCl3 (10.0 mL). The resulting mixture
was refluxed for 12 h. After cooling to r.t. the reaction mixture was added with Et2O (5 mL). The
white precipitate was filtered and washed with Et2O. The crude product was sufficiently pure to
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be used without further purification. Yield: 65%; mp = 180ꢀ183 °C. H NMR (CDCl3, ppm):
5.07 (s, 2H); 7.28ꢀ7.30 (m, 1H); 7.33ꢀ7.38 (m, 3H); 7.47ꢀ7.49 (m, 2H); 7.83 (d, 1H, J=4.8 Hz);
8.76 (d, 1H, J=3.6 Hz). 13C NMR (CDCl3, ppm): 176.95, 137.83, 137.04, 132.48, 131.78,
130.39, 129.76, 129.27, 128.91, 40.04.
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Naphth-2-ylmethyl thiophene-2-carbimidothioate (10). Compound 10 was obtained from
compound 28 (0.300 g, 2.10 mmol) and 2ꢀ(bromomethyl)naphthalene (0.460 g, 2.10 mmol)
following the same procedure described for 9. The crude product was sufficiently pure to be used
without further purification. Yield: 55%; mp = 200ꢀ201 °C; lit. ref. n.35: mp = 198ꢀ199 °C.13C
NMR (CDCl3, ppm): 176.97, 137.89, 137.12, 133.42, 133.29, 132.58, 130.46, 129.40, 129.32,
129.08, 128.11, 127.88, 126.90, 126.82, 126.81, 40.48.
Phenyl thiophene-2-carbimidothioate hydrobromide (11). Compound 11 was obtained from
2ꢀthiophenecarbonitrile 30 (1.00 g, 9.16 mmol) and thiophenol (0.93 mL, 9.16 mmol) in Et2O
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(1mL) following the same procedure described for 6. Yield: 63%; mp = 220ꢀ221°C. H NMR
(CDCl3, ppm): 7.36 (dd, 1H, J=5.2,4.8 Hz ); 7.63ꢀ7.66 (m, 4H ); 7.70ꢀ7.73 (m, 1H ); 7.98 (dd,
1H, J = 5.2, 1.2 Hz ); 8.10 (br s, 1H, NH); 8.99 (dd, 1H, J= 4.8, 1.2 Hz). 13C NMR (CDCl3,
ppm): 178.05, 139.25, 139.18, 136.05, 133.82, 132.03, 130.85, 130.72, 121.22.
N-Benzylbenzothioamide (12) . A mixture of Nꢀbenzylbenzamide 22 (0.300 g, 1.40 mmol) and
Lawesson’s reagent (0.679 g, 1.70 mmol) in 15.0 mL of dry THF was stirred at room
temperature for 12 h (TLC analysis). Then, the organic solvent was evaporated to dryness. The
resulting solid was washed with a 5% solution of NaHCO3 and then extracted with AcOEt . The
organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduced
pressure. The product was finally purified by flash chromatography (petroleum ether 60ꢀ80
°C/AcOEt=6/4 as eluent).Yield: 90%; mp = 78ꢀ80 °C; lit. ref. n.36: mp = 84ꢀ85 °C.
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