Synthesis and Pharmacological Evaluation of Novel Adenine-Hydrogen Sulfide Slow Release Hybrids Designed as Multitarget Cardioprotective Agents

Article abstract of DOI:10.1021/acs.jmedchem.5b01223

This work deals with the design, synthesis, and evaluation of the cardioprotective properties of a number of novel hybrid compounds combining the adenine nucleus with a suitable H₂S slow-releasing moiety, coupled via a stable ether bond. The H₂S release rate of the hybrids and their ability to increase cGMP were estimated in vitro. The most promising derivatives 4 and 11, both containing 4-hydroxythiobenzamide moiety as H₂S donor, were selected for further in vivo evaluation. Their ability to release H₂S in vivo was recorded using a new fully validated UPLC-DAD method. Both compounds reduced significantly the infarct size when administered at the end of sustained ischemia. Mechanistic studies showed that they conferred enhanced cardioprotection compared to adenine or 4-hydroxythiobenzamide. They activate the PKG/PLN pathway in the ischemic myocardium, suggesting that the combination of both pharmacophores results in synergistic cardioprotective activity through the combination of both molecular pathways that trigger cardioprotection.

Full text of DOI:10.1021/acs.jmedchem.5b01223

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Article
SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF NOVEL
ADENINE-HYDROGEN SULFIDE SLOW RELEASE HYBRIDS
DESIGNED AS MULTI-TARGET CARDIOPROTECTIVE AGENTS
Nikolaos Lougiakis, Andreas Papapetropoulos, Evagelos Gikas, Spyridon Toumpas, Panagiotis
Efentakis, Rudolf Wedmann, Anastasia Zoga, Zhongmin Zhou, Efstathios K Iliodromitis, Alexios-
Leandros Skaltsounis, Milos R. Filipovic, Nicole Pouli, Panagiotis Marakos, and Ioanna Andreadou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01223 • Publication Date (Web): 26 Jan 2016
Downloaded from http://pubs.acs.org on January 28, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF NOVEL
ADENINE-HYDROGEN SULFIDE SLOW RELEASE HYBRIDS DESIGNED
AS MULTI-TARGET CARDIOPROTECTIVE AGENTS
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a
a
a
Nikolaos Lougiakis, Andreas Papapetropoulos, Evangelos Gikas, Spyridon
a
a
b
a,c
Toumpas, Panagiotis Efentakis, Rudolf Wedmann, Anastasia Zoga, Zhongmin
a
c
d
Zhou, Efstathios K. Iliodromitis, AlexiosꢀLeandros Skaltsounis, Milos R.
b
a*
a
a*
Filipovic, Nicole Pouli, Panagiotis Marakos, Ioanna Andreadou
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, National and
Kapodistrian University of Athens, Greece
b
Department of Chemistry and Pharmacy, FriedrichꢀAlexander University of
ErlangenꢀNuremberg, Erlangen, Germany
c
Second University Department of Cardiology, Medical School, Attikon General
Hospital, National and Kapodistrian University of Athens, Greece
d
Department of Pharmacognosy & Natural Products Chemistry, Faculty of
Pharmacy, National and Kapodistrian University of Athens, Greece
Address for correspondence:
Ioanna Andreadou, Ph.D, National and Kapodistrian University of Athens, Faculty of
Pharmacy, Panepistimiopolis Zografou, Athens 15771, GREECE; tel:+30 210
7
274827; fax:+30 210 7274747; eꢀmail: jandread@pharm.uoa.gr
Nicole Pouli, National and Kapodistrian University of Athens, Faculty of Pharmacy,
Panepistimiopolis Zografou, Athens 15771, GREECE; tel:+30 210 7274185; fax:+30
2
10 7274747; eꢀmail: pouli@pharm.uoa.gr
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Abstract
This work deals with the design, synthesis and evaluation of the cardioprotective
properties of a number of novel hybrid compounds combining the adenine nucleus
with a suitable H S slowꢀreleasing moiety, coupled via a stable ether bond. The H S
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release rate of the hybrids and their ability to increase cGMP were estimated in vitro.
The most promising derivatives 4 and 11, both containing 4ꢀhydroxythiobenzamide
moiety as H
2
S donor, were selected for further in vivo evaluation. Their ability to
release H S in vivo was recorded using a new fully validated UPLCꢀDAD method.
2
Both compounds reduced significantly the infarct size, when administered at the end
of sustained ischemia. Mechanistic studies showed that they conferred enhanced
cardioprotection compared to adenine or 4ꢀhydroxythiobenzamide. They activate the
PKG/PLN pathway in the ischemic myocardium suggesting that the combination of
both pharmacophores results in synergistic cardioprotective activity through the
combination of both molecular pathways that trigger cardioprotection.
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INTRODUCTION
Acute myocardial infarction (AMI) is the main cause of morbidity and
mortality in the world. Reperfusion techniques, such as thrombolysis and coronary
artery bypass, can reꢀestablish the coronary blood flow, resulting in a significant
decrease of the ischemiaꢀrelated damage. However, reperfusion itself contributes to
the total injury and causes a number of deleterious effects to the ischemic myocardial
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1
tissue, a procedure known as reperfusion injury. This irreversible damage refers to
cell death associated with prolonged ischemia and may be prevented by an
intervention applied at the time of reperfusion. In that respect, Zhao et al. first
proposed that ischemic postconditioning (PostC), whereby brief episodes of
ischemia/reperfusion applied at the onset of reperfusion, could attenuate reperfusion
2
injury. Although PostC has been shown to be successful in preclinical models, it has
failed to translate into therapeutically useful cardioprotective strategies that impact
3
everyday clinical practice. Thus, the administration of pharmacological PostC
mimetics may provide some clinical advantages. The use of a safe drug as a PostC
medication would be fairly easy, provided that it could be available in an intravenous
formulation and can be administered effectively, in order to cover the time window
4
for protection against lethal reperfusion injury. To achieve this goal different
pharmacological manipulations of the cellular events have been proposed based on
5,6
the advances in our understanding of the involved pathogenetic mechanisms.
Nonlethal ischemia induces the production of endogenous autacoids, such as
adenosine, bradykinin and opiods, which upon binding to their corresponding
receptors initiate cardioprotective signaling pathways, leading to subsequent
7
inhibition of the mitochondrial permeability transition pore (mPTP) opening. mPTP
8
opening, which plays a key role in myocardial necrosis at reperfusion, is regulated by
2
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several factors, including Ca overload, mitochondrial matrix pH, decrease of
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membrane potential, and excessive production of reactive oxygen species (ROS).
Thus, selective inhibition of mPTP opening appears to be a promising therapeutic
strategy against myocardial ischemiaꢀreperfusion injuries.
Although a number of studies have demonstrated the cardioprotective effects
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of endogenous H S in myocardial ischemia/reperfusion injury,
there exist limited
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reports concerning the cardioprotective effects of exogenous administration of H S in
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models of ischemia/reperfusion. We have recently shown that administration of
sodium hydrogen sulfide (NaHS, a salt of H
cGMP/PKG/phosholamban (PLN) pathway thereby preventing mPTP opening.
However, the exposure of biological systems to inorganic H S salts, causes a burst of
2
S) induces cardioprotection via a
12
2
H
2
S that does not recapitulate the low level of continuous production of H
occurs in vivo. For these reasons, the discovery of pharmacologically useful
precursors for endogenous H S synthesis or, alternatively the use of H Sꢀslow
releasing compounds seems to be a convenient and compelling strategy.
established medicinal chemistry approach in this field deals with the covalent
incorporation of a H S donating group into the structure of a biologically active
derivative. Thus, a number of H S releasing moieties have been developed, including
morpholinꢀ4ꢀium 4ꢀmethoxyphenylmorpholinophosphinodithioate (GYY4137,
2
S that
2
2
1
3,14
An
2
2
a
water soluble derivative of the Lawesson’s reagent), arylthiobenzamides, 4ꢀ
hydroxythiobenzamide (4ꢀOHꢀTBZ), anethole dithiolethione (ADT) and its
15,16
metabolite 5ꢀ(4ꢀhydroxyphenyl)ꢀ3Hꢀ1,2ꢀdithioleꢀ3ꢀthione (ADTꢀOH).
A number
of hybrid compounds are currently under intensive investigation possessing a broad
1
7
2
spectrum of activities, and in particular some H Sꢀreleasing nonsteroidal antiꢀ
inflammatory drug (NSAID) derivatives have been reported to display increased
14, 15, 18
effectiveness and reduced toxicity, when compared to their parent drugs.
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However, there are limited novel H S releasing molecules in the literature,
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acting either as slow, or controllable releasers, with protective effects against
ischemia/ reperfusion injury. To the best of our knowledge there are no reports of
hybrids possessing H Sꢀreleasing properties that when administered at a clinical
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setting (late at ischemia and during reperfusion) limit myocardial infarction.
On the other hand, an increasing body of evidence has shown that adenosine
receptors are involved in endogenous protective mechanisms against myocardial
2
0
ischemia/reperfusion injury. Exogenously administered adenosine and its selective
agonists reduced infarct size, but this benefit was not translated into improved clinical
21,22
outcome.
Additionally, the ability of intracoronary adenosine infusion
immediately before coronary stenting to limit infarct size has been tested in clinical
23,24
trials with disappointing results.
These controversial data might be attributed to
the use of inadequate doses of the drug, which are not high enough to overcome
25
adenosine’s extremely rapid metabolism in red blood cells and endothelial cells.
Furthermore, the high dose required to exert cardioprotective effects, can be
associated with severe hypotension and bradycardia, due to vasodilatory and negative
2
5,26
chronotropic effects of the drug.
These hemodynamic consequences limit the
26
usefulness of adenosine, as a cardioprotective agent in humans.
Having in mind the beneficial effects of both adenosine and hydrogen sulfide
in the cardiovascular system during ischemia/reperfusion, we hypothesized that H Sꢀ
2
releasing derivatives of adenosine could display synergistic effects, exerted by both
pharmacophore components. We have thus designed a series of new hybrid
compounds, which were evaluated as potential cardioprotective agents in vitro and in
vivo. In this respect, adenosine, as well as the nonꢀnucleoside analogue of adenine, 9ꢀ
(4ꢀhydroxybutyl)adenine, were coupled with 4ꢀOHꢀTBZ and ADTꢀOH (Figure 1) via
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the chemically and metabolically stable ether bond. The absence of a free 5’ꢀhydroxyl
group could reduce the rapid metabolism of adenosine in blood, mainly caused by the
action of adenosine kinase and adenosine deaminase. In the case of the corresponding
butyl analogues, the absence of a ribosyl moiety is expected to provide even more
stable derivatives.
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In order to assess the effectiveness of these compounds in releasing H S in
vivo, a UPLCꢀDAD analytical method has been developed and validated permitting
the measurement of thiosulfate circulating levels, upon the administration of the new
compounds. Although the accurate and reliable assessment of free H S concentrations
2
in vivo could provide critical information about its role, its precise measurement in
multiple biological matrices is controversial, due to its oxidation in mitochondria to
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thiosulfate and sulfate by specific enzymes. Since most of the sulfate is derived from
2
H Sꢀindependent sources, such as cysteine oxidation, thiosulfate is considered the
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most specific marker of H S metabolism.
2
RESULTS AND DISCUSSION
Chemistry. 5ꢀ(4ꢀHydroxyphenyl)ꢀ3Hꢀ1,2ꢀdithioleꢀ3ꢀthione (ADTꢀOH) was
synthesized in two steps, according to a literature procedure starting from anethole.
Thus, anethole was initially treated with sulfur in dimethylacetamide to provide the
intermediate 5ꢀ(4ꢀmethoxyphenyl)ꢀ3Hꢀ1,2ꢀdithioleꢀ3ꢀthione, which was finally
29
demethylated upon heating with pyridine HCl.
Concerning the preparation of the target adenosine analogues, adenosine was
converted to the corresponding 2’,3’ꢀisopropylidene derivative 2 (Scheme 1),
followed by ether formation under Mitsunobu conditions, upon treatment with ADTꢀ
OH or commercially available 4ꢀhydroxythiobenzamide, in the presence of DIAD and
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PPh
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. The intermediate ethers 3 and 5 were then deprotected under acidic conditions,
providing the target compounds 4 and 6.
On the other hand, commercially available adenine (7, Scheme 2) was
converted to the corresponding 9ꢀ(4ꢀhydroxybutyl)acetate 8 upon treatment with 4ꢀ
bromobutylacetate under basic conditions and the isolated Nꢀ9 isomer was
subsequently treated with methanolic ammonia at room temperature, providing the
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alcohol 9. Mitsunobu type reaction of 9 with ADTꢀOH, 4ꢀOHꢀTBZ, or 4ꢀ
hydroxybenzamide, resulted into the target compounds 10ꢀ12.
In vitro hydrogen sulfide release evaluation. The newly synthesized
compounds 4, 6, 10 and 11, together with the intermediate isopropylideneꢀprotected
nucleosides 3 and 5 were tested using an in vitro assay in order to evaluate their
ability to release hydrogen sulfide. The methylene blue method is the most commonly
3
1,32
reported one to measure hydrogen sulfide in biological samples.
It is based on the
colorimetric determination of methylene blue dye, resulting from the reaction of
3
+
sulfide with N,Nꢀdimethylꢀpꢀphenylenediamine sulfate in the presence of Fe as an
oxidizing agent. In this assay all compounds demonstrated a concentrationꢀdependent
H
2
S release. Nevertheless, the 4ꢀOHꢀTBZ derivatives 3, 4 and 11 produced high
amounts of H S, whereas the isopropylidene derivative 5 as well as the 5’ꢀadenosine
and adenine ethers 6 and 10 respectively, all bearing the ADTꢀOH substitution,
released lower levels of H S (Figure 2A).
To further confirm the H Sꢀdonating ability of these compounds we used
amperometric method with selective H S electrode. All compounds showed slow and
steady H S release. The representative electrode trace of one of the compounds
2
2
2
2
2
(compound 4) is shown in Figure 2. Compound 4 showed biphasic H S release: an
2
instantaneous release of small H
2
S amounts was noticeable when DMSO stock
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solution was added to buffer, followed by very slow and steady H S release (Figure
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B). Control electrode trace of 10 µM Na S showed completely opposite kinetics: a
2
constant decay due to the fact that the whole amount of sulfide ended up as H
which then slowly oxidized and got physically removed from the system (Figure 2C).
Addition of compound 12, which has no H S donating properties, led to no change in
electrode response (Figure 2C). Interestingly, addition of 1 mM glutathione (GSH)
slowed down the H S release from compound 4, suggesting an interaction between
2
S,
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the two molecules (Figure 2D).
We should mention that the results obtained with the H S electrode and the
2
methylene blue method are qualitatively similar. In both cases, when a low
concentration of compound 4 was used, no H
concentration (150 ꢁM for the methylene blue and 100 ꢁM for the electrode
measurements) yielded measurable H S amounts.
Characterization of H S release mechanism. To elucidate the possible
2
S could be detected, while the higher
2
2
2
mechanism of H S release by this class of compounds, we used timeꢀresolved UHRꢀ
ESIꢀTOFꢀMS. Stock solutions of compound 4 in DMSO, were diluted with 20 mM
ammonium carbonate buffer and spectra were continuously recorded during 40 min
(Figure 3A). The presence of m/z 387.1414 peak which corresponds to the
+
[
C H N O +H] (calculated m/z 387.1411) (Figure 3B), as well as of m/z 371.1464
1
7
18
6
5
+
peak which corresponds to the [C17
18 6 4
H N O +H] (calculated m/z 371.1462) were
immediately noticeable (Figure 3C). This suggests that possible mechanism for H
2
S
release could be a slow hydrolysis of thioamide bond. Furthermore, the main peak
+
([4+H] , observed at m/z 403.1184, calculated 403.1183) showed time dependent
decay (Figure 3D). Assuming that the initial peak intensity corresponds to the amount
of added compound 4, and by plotting the changes in peak intensity vs time (Figure
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3E), we calculated the rate of H S release from 100 µM of compound 4 to be 0.8±0.1
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µM/min at pH 7.4 (21 °C). Interestingly, when glutathione (GSH) was mixed with
compound 4, an immediate formation of small amounts of glutathione persulfide
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could be observed at m/z 340.0636 (calculated m/z 340.0632) (Figure 3F), and at m/z
+
6
47.1475 corresponding to the cluster of [GSH + GSSH + H] (calculated 647.1470,
not shown). This suggests that under physiological conditions, in parallel to a direct
S release this class of compounds could lead to the formation of low molecular
H
2
weight persulfides, which could explain the slower H S release observed in Figure
2
2D. GSSH has been recently suggested to be one of the main mediators of H2S
33,34
biological effects leading to transꢀpersulfidation of protein targets.
Considering
mM concentration of GSH in the cells, it is tempting to speculate that biological
effects of this type of H S donors will partly, if not largely, derive from the formation
2
of GSSH and subsequent protein persulfidation.
In vitro evaluation of cGMP measurements. As we have previously shown,
2
H S increases cGMP by inhibiting phosphodiesterase (PDE) and by enhancing eNOS
35,36
activity.
Many studies have also showed a consistent protective effect of
37ꢀ39
interventions aimed to increase cGMP in reperfused myocardium.
Thus, in order
to assess the biological activity of the novel hybrids we investigated their ability to
increase cGMP, by measuring the levels of cGMP in cultured cells. The compounds
2
that yielded more H S in the methylene blue assay, namely 3, 4 and 11, also caused a
substantial increase in cGMP (Figure 4). It should be noted that 9ꢀ(4ꢀ
hydroxybutyl)adenine as well as adenosine, did not increase cGMP levels (data not
shown). The cGMP accumulation in response to the hybrid molecules was lower than
that of the parent H Sꢀreleasing moiety (4ꢀOHꢀTBZ), suggesting that the adenine
2
nucleus contributes in the stabilization of the molecule in the cellular milieu and
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reduces the rate of H
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S generation. Based on these results and taking under
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consideration that compounds that could release an adequate amount of H S may
possibly have a better pharmacological effect, compounds 4 and 11, which clearly
possess the better profile as H S donors, were chosen for further evaluation to the in
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vivo experiments.
In vivo evaluation of the cardioprotective activity of the tested
compounds. Recent studies with animal models suggested that H
2
S can protect
1
0,16,19
cardiovascular system against myocardial ischemia/reperfusion injury.
The
selected compounds 4 and 11 were thus administered in anesthetized rabbits before
reperfusion in a single dose. We have chosen a dose which is 20 fold lower than the
1
2
corresponding dose of NaHS and does not impair the hemodynamic parameters. In
pilot experiments we had determined the optimal doses for compounds 4 and 11.
Herein we provide results with compound 11 in a double dose (11dd). The
experimental protocol is illustrated in Figure 5. No significant differences were
observed on heart rate and mean arterial blood pressure among all studied groups,
with the exception of group 11dd, where a significant reduction in mean blood
pressure was observed immediately after the administration of the compound
(characteristic and hemodynamic variables for all studied rabbit groups are presented
in Table 1). Thus, dose response estimation was selected on the basis of the dose that
does not impair the hemodynamic parameters.
The effect of the tested compounds on infarct size reduction is compared with
the control group as well as with the PostC and NaHS groups, as positive controls. No
significant differences were detected in the areas at risk among the studied groups
(Figures 6B, 7B, 8B). Application of PostC (as a positive control) limited infarct size
compared to Control (24.35±1.3% vs 47.9±0.7%, P<0.05), as expected (Figure 6A).
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NaHS reduced the infarct size compared to Control group (16.7±2.0% vs 47.9±0.7%,
P<0.05) and to a greater extent than PostC (P<0.05, Figure 6A). Compound 11
reduced significantly the infarct size compared to the Control and PostC groups
(16.6±0.3% vs 47.9±0.7% and 24.35±1.3% respectively, P<0.05, Figure 6A).
10
11
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Although administration of the double dose of compound 11 reduced the infarct size
compared to the Control group (26.7±1.6, P<0.05, Figure 6A), this reduction was in a
lower extent than the one observed with the administration of the single dose of 11
(P<0.05 vs 11). The significant reduction of the mean blood pressure upon
administration of double dosage of compound 11 could explain the efficacy
limitation, due to hemodynamic instability. Compound 4 reduced infarct size
significantly when compared to the Control group (20.1±0.9%, vs 47.9±0.7%,
P<0.05) and to a similar extent with that of NaHS and PostC (Figure 6A). These
results demonstrate that the adenosine analogue 4, as well as the adenine derivative
11, both bearing the 4ꢀOHꢀTBZ moiety, are powerful agents in reducing the
myocardial infarct size when administered at a clinical based protocol (at the end of
sustained ischemia). The novel compounds possessed similar cardioprotective effects
compared to NaHS, although at doses estimated to be 20ꢀfold lower.
2
In order to investigate if the compounds exhibit H Sꢀmediated cardiac
protection in ischemia/reperfusion injury we have also administered in anesthetized
rabbits the carboxamide 12, which does not possess H S releasing properties and
2
examined its effect on infarct size. We found that compound 12 reduced the infarct
size compared to the control group (26.6±1.0%, P<0.05 vs Control, Figure 7A),
probably through activation of the adenosine receptors. However, the %I/R achieved
upon administration of compound 11 in terms of statistical significance was lower
than that of 12 (P<0.05). Additionally, administration of 9ꢀ(4ꢀhydroxybutyl)adenine
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(9), resulted in a similar biological effect (32.4±2.8%, P<0.05 vs Control and 11,
Figure 7A). These results reveal that although both 9 and 12 have a limiting effect on
infarct size, compound 11 showed significantly improved profile, due to the presence
of the H S releasing group, indicating that this moiety is crucial for enhanced
2
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cardioprotection. To further verify the aforementioned result, we coꢀadministered
compound 12 with NaHS and observed reduction of the infarct size compared to the
Control group (22.3±2.5%, P<0.05 vs Control, Figure 7A); however although the %
I/R was higher than the one of compound 11, this difference did not reach statistical
significance. We should point out that compound 11 has the advantage that it can
reduce very significantly the infarct size when it is administered as a single bolus
dose, without affecting hemodynamic parameters, whereas, NaHS should be
administered with continuous infusion and in 20 fold higher concentration compared
to 11, in order to establish infarction limitation with hemodynamic stability.
With the aim of further evaluating the contribution of the two components of
the hybrid compounds 4 and 11 to the final cardioprotective effect, we coꢀ
administered the tested compounds with the non selective inhibitor of adenosine
receptors 8ꢀ(pꢀsulfophenyl)theophylline (SPT). Effectiveness of SPT in inhibiting
40
adenosine receptors in cardioprotection has been demonstrated. As shown in Figure
8
A, the administration of SPT did not inhibit the infarct size limiting effects of 11 and
4 (16.9±1.8% and 18.3±0.7%, respectively, P=NS vs 11, 4), indicating that the
adenine moiety may not be the main determinant for cardioprotection. However, in
order to confirm that the adenine moiety triggers cardioprotection through adenosine
2
receptors, we coꢀadministered the carboxamide 12, which does not possess H S
releasing properties, with SPT. As expected, an abrogation of infarct size of
compound 12 was observed when adenosine receptors are inhibited (41.8±1.7 %,
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P<0.05 vs 12), indicating that the adenine nucleus contributes to cardioprotection
(Figure 8A).
In order to investigate whether the hybrid molecules confer enhanced
10
11
12
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cardioprotection when compared to 4ꢀOHꢀTBZ alone, we administered compound 4ꢀ
OHꢀTBZ in anesthetized rabbits and we observed that it reduced the infarct size
significantly compared to the Control group (24.9±0.7%, P<0.05, Figure 7A),
although to a lesser extent than compound 11 (P<0.05 vs 11), indicating that the
adenine moiety is also essential for enhanced cardioprotective activity. Taken
together, the above results strengthen our conclusion that the hybrid molecules have
enhanced cardioprotective activity when compared to adenine or to 4ꢀOHꢀTBZ. We
should also mention that we show herein for first time that 4ꢀOHꢀTBZ reduced infarct
size in vivo.
It has been proposed that the activation of mitochondrial potassium channels
(
mitoKATP) is a powerful mechanism of cardioprotection of H S against
2
10
ischemia/reperfusion. However, we have recently showed that mitoKATP channels
do not mediate H Sꢀinduced cardioprotection in rabbits in a protocol similar to the
current study, and H S triggers cardioprotection through a cGMP/PKG/PLN
2
12
2
pathway. Since the goal of the present study was to combine in one molecule two
pharmacophores, we focused in the concomitant activation of adenosine receptors and
the cGMP/PKG/PLN pathway. Compounds 4 and 11 increase cGMP levels in
cultured cells, therefore we focused further on PKG/PLN pathway.
One of the major biological actions of cGMP is the activation of cGMPꢀ
dependent protein kinases G (PKG) I and II and we have shown that exposure of
endothelial cells or vessels to NaHS led to increased cGMP accumulation and
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activation of PKG, as suggested by an increase in VASP phosphorylation, a surrogate
3
6,41
marker of its activity.
In line with this finding, we noted that VASP
phosphorylation was increased in the ischemic myocardium of both rabbits and mice
12
after the administration of NaHS. In the present study, we confirmed our previous
findings and we observed that both compounds 4 and 11 increased significantly the
phosphorylation of VASP, compared to the Control group, indicating that the novel
hybrid molecules trigger cardioprotection via a cGMP/PKG pathway (Figure 9A).
We have also identified phospholamban (PLN) as a downstream target of
0
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2+
PKG; PLN once phosphorylated reduces free intracellular Ca concentration by
2+
dissociating from SERCA allowing it to pump Ca back into the sarcoplasmic
41
reticulum, limiting the damage from hypercontracture and mPTP opening. We
observed that both compounds 4 and 11 activate PLN in the ischemic myocardium at
th
the 10 min of reperfusion, compared to Control group (Figure 9B). Therefore, the
combination of two pharmacophores into the new hybrid derivatives results in
synergistic cardioprotective activity through the combination of both molecular
pathways that trigger cardioprotection (Figure 10).
Determination of plasma levels of thiosulfates with the UPLC-DAD
method. We have then attempted to determine if the above mentioned hybrids could
actually release H S in vivo. Thus, circulating levels of thiosulfates, the major
2
metabolite of H S, were determined using a new UPLCꢀDAD analytical method,
2
42
which is a modification of the method of Chwatko et al. Briefly, the determination
of thiosulfate in plasma is based on the precolumn derivatization of analyte, based on
its ability to oxidize 2ꢀchloroꢀ1ꢀmethylquinolinium tetrafluoroborate (CMQT) to 1ꢀ
methylꢀ2ꢀthioquinolone (MTQ) at pH 2. It should be noted that reaction of CMQT
with thiosulfates in the presence of sulfites in low pH (~2) leads to the selective
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reaction of the former, whereas reaction at physiological pH (7.4) produces response
from both molecules. The developed analytical method has been applied to plasma
samples, employing a simple and fast sample cleanup procedure based on protein
precipitation, whereas the analysis time was kept below 2 min, affording a very fast
sample turnover. This methodology could potentially be used in pharmacokinetic
studies due to its rapidity, without compromise of resolution and the overall analytical
performance. The use of an internal standard (αꢀnaphthol) enhances the reliability of
the developed methodology. The method has been fully validated according to the
ICH Q2R1 and EMA guidelines on bioanalytical method validation. The
methodology has been found to be suitable for the determination of thiosulfate in
10
11
12
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2
plasma as it demonstrated adequate linearity (R =0.994) for the tested range of 0.25ꢀ
ꢀ1
2
0 ꢁmol L , whereas with recovery and accuracy values well between the required by
the guidelines perquisites. The sensitivity of the method has been determined to be
ꢀ1
0.233 ꢁmol L as expressed by the LOQ value. The results of the validation study are
provided in Supplementary Data File, whereas a representative chromatogram of a
thiosulfate positive plasma sample is shown in Figure 11.
CONCLUSION
The results of the present study highlight the discovery of two novel hybrid
compounds derived from the combination of adenosine as well as 9ꢀ(4ꢀ
hydroxybutyl)adenine with the hydrogen sulfide donor 4ꢀOHꢀTBZ. The new hybrids
act as H
administered during myocardial ischemia. To the best of our knowledge this is the
first study to design and utilize a combined H S/adenosine strategy in a single
2
S slow releasers and reduce significantly the myocardial infarct size when
2
molecule that is based on the exploitation of two distinct signaling pathways of
cardioprotection. The new compounds have similar activity to that of NaHS, although
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they were administered in a considerably lower dose. Our study provides the basis for
2
further development and testing of H S donors in heart disease.
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EXPERIMENTAL SECTION
Chemistry
1
Melting points were determined on a Büchi apparatus and are uncorrected. Hꢀ
NMR spectra and 2ꢀD spectra were recorded on a Bruker Avance 400 instrument,
13
whereas CꢀNMR spectra were recorded on a Bruker AC 200 spectrometer in
1
13
deuterated solvents and were referenced to TMS (δ scale). The signals of H and C
1
1
spectra were unambiguously assigned by using 2D NMR techniques: H H COSY,
NOESY, HMQC and HMBC. Mass spectra were recorded with a LTQ Orbitrap
Discovery instrument, possessing an Ionmax ionization source. Flash chromatography
was performed on Merck silica gel 60 (0.040ꢀ0.063 mm). Analytical thin layer
chromatography (TLC) was carried out on precoated (0.25 mm) Merck silica gel Fꢀ
2
54 plates. Elemental analyses were performed on a PerkinꢀElmer PE 240C Elemental
Analyzer (Norwalk, CT, U.S.A.) and were within ± 0.4% of the theoretical values,
suggesting purity >95%.
General procedure for the synthesis of 3, 5, 10-12. Diisopropyl
azodicarboxylate (295 µL, 1.5mmol) was added to an iceꢀcooled solution of
triphenylphosphine (393 mg, 1.5 mmol) in dry tetrahydrofuran (15 mL), under argon
o
and the mixture was stirred at 0 C for 15 minutes. Then, compound 2 (1 mmol) or 9
(1 mmol) was added and stirring was continued at 0 °C for 5 more minutes, followed
by addition of 5ꢀ(4ꢀhydroxyphenyl)ꢀ3Hꢀ1,2ꢀdithioleꢀ3ꢀthione (1 mmol), or 4ꢀ
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hydroxythiobenzamide (1 mmol), or 4ꢀhydroxybenzamide (1 mmol). The resulting
mixture was stirred at room temperature for 48 hours and the crude product was dryꢀ
packed and purified by flash column chromatography to provide pure ethers 3, 5, 10-
1
2, respectively.
10
11
12
13
14
15
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2
’,3’-O-isopropylidene-5’-O-(4-carbamothioylphenyl)adenosine (3). This
compound was prepared according to the general procedure described above, using
’,3’ꢀisopropylideneadenosine and 4ꢀhydroxythiobenzamide. Purification was
2
effected by flash chromatography using a mixture of chloroform / methanol (from
1
1
00/3 up to 100/6) as the eluent. Yield: 48%. M.p. 156ꢀ7 °C (EtOAc/Et O). HꢀNMR
2
(
400 MHz, DMSOꢀd
6 3 3
) δ 1.36 (s, 3H, CH ), 1.57 (s, 3H, CH ), 4.21 (dd, 1H, Hꢀ5’,
J=10.45 Hz + 5.79 Hz), 4.28 (dd, 1H, Hꢀ5’, J=10.45 Hz + 4.63 Hz), 4.55 (m, 1H, Hꢀ
4
’), 5.14 (m, 1H, Hꢀ3’), 5.54 (m, 1H, Hꢀ2’), 6.23 (d, 1H, Hꢀ1’, J=2.37 Hz), 6.90 (d,
H, Hꢀ2’’+Hꢀ6’’, J=8.91 Hz), 7.32 (brs, 2H, D O exch., adenineꢀNH ), 7.91 (d, 2H,
2
2
2
Hꢀ3’’+Hꢀ5’’, J=8.91 Hz), 8.18 (s, 1H, Hꢀ2), 8.31 (s, 1H, Hꢀ8), 9.31 (brs, 1H, D O
2
13
exch., NHꢀthiocarboxamide), 9.64 (brs, 1H, D
NMR (50 MHz, DMSOꢀd ) δ 25.22 (CH ), 27.03 (CH
3.39 (Cꢀ2’), 84.04 (Cꢀ4’), 89.50 (Cꢀ1’), 113.36 (Cꢀisopropylidene), 113.49 (Cꢀ
’’+Cꢀ6’’), 119.07 (Cꢀ5), 129.42 (Cꢀ3’’+Cꢀ5’’), 131.79 (Cꢀ4’’), 139.55 (Cꢀ8), 148.90
2
O exch., NHꢀthiocarboxamide). Cꢀ
6
3
3
), 67.95 (Cꢀ5’), 81.44 (Cꢀ3’),
8
2
(
Cꢀ4), 152.78 (Cꢀ2), 156.12 (Cꢀ6), 160.59 (Cꢀ1’’), 198.55 (C=S). HRꢀMS (ESI) m/z:
+
Calcd for C20
H
23
N
6
O
4
S: [M1+H] =443.1496, found 443.1495. Anal. Calcd for
20 22 6 4
C H N O S: C, 54.29; H, 5.01; N, 18.99. Found: C, 54.13; H, 4.89; N, 19.08.
2
’,3’-O-isopropylidene-5’-O-[4-(3-thioxo-3H-1,2-dithiol-5-
yl)phenyl]adenosine (5). This compound was prepared according to the general
procedure described above, using 2’,3’ꢀisopropylideneadenosine and 5ꢀ(4ꢀ
hydroxyphenyl)ꢀ3Hꢀ1,2ꢀdithioleꢀ3ꢀthione. Purification was effected by flash
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5
5
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6
chromatography using a mixture of dichloromethane / methanol (from 100/1 up to
1
1
00/4) as the eluent. Yield: 45%. M.p. 193 °C (EtOAc/Et
DMSOꢀd ) δ 1.36 (s, 3H, CH ), 1.57 (s, 3H, CH
.77 Hz), 4.33 (dd, 1H, Hꢀ5’, J=10.50 Hz + 4.55 Hz), 4.56 (m, 1H, Hꢀ4’), 5.15 (m,
1H, Hꢀ3’), 5.55 (m, 1H, Hꢀ2’), 6.24 (d, 1H, Hꢀ1’, J=2.51 Hz), 7.03 (d, 2H, Hꢀ2’’+Hꢀ
2
O). HꢀNMR (400 MHz,
6
3
3
), 4.25 (dd, 1H, Hꢀ5’, J=10.50 Hz +
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
6
3
’’, J=8.90 Hz), 7.32 (brs, 2H, D
2
O exch., NH
2
), 7.75 (s, 1H, Hꢀ5’’’), 7.84 (d, 2H, Hꢀ
13
’’+Hꢀ5’’, J=8.90 Hz), 8.18 (s, 1H, Hꢀ2), 8.31 (s, 1H, Hꢀ8). CꢀNMR (50 MHz,
DMSOꢀd
3.94 (Cꢀ4’), 89.47 (Cꢀ1’), 113.36 (Cꢀisopropylidene), 115.47 (Cꢀ2’’+Cꢀ6’’), 119.07
(Cꢀ5), 124.07 (Cꢀ4’’), 128.99 (Cꢀ3’’+Cꢀ5’’), 134.31 (Cꢀ5’’’), 139.57 (Cꢀ8), 148.88
6 3 3
) δ 25.21 (CH ), 27.02 (CH ), 68.11 (Cꢀ5’), 81.37 (Cꢀ3’), 83.33 (Cꢀ2’),
8
(Cꢀ4), 152.76 (Cꢀ2), 156.10 (Cꢀ6), 161.39 (Cꢀ1’’), 173.63 (Cꢀ1’’’), 214.84 (Cꢀ4’’’).
+
HRꢀMS (ESI) m/z: Calcd for C22
H
22
N
5
O
4
S
3
: [M1+H] =516.0828, found 516.0825.
Anal. Calcd for C22
N, 13.29.
21 5 4 3
H N O S : C, 51.25; H, 4.11; N, 13.58. Found: C, 51.41; H, 4.17;
9-{4-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl]butoxy}adenine (10). This
compound was prepared according to the general procedure described above, using 9ꢀ
(
4ꢀhydroxybutyl)adenine
and
5ꢀ(4ꢀhydroxyphenyl)ꢀ3Hꢀ1,2ꢀdithioleꢀ3ꢀthione.
mixture of
Purification was effected by flash chromatography using
a
dichloromethane / methanol (from 100/1 up to 100/4) as the eluent. Yield: 39%. M.p.
1
233 °C (THF). HꢀNMR (400 MHz, DMSOꢀd
6
) δ 1.71 (m, 2H, Hꢀ3’), 1.97 (m, 2H, Hꢀ
2
2
2
’), 4.09 (t, 2H, Hꢀ4’, J=6.38 Hz), 4.22 (t, 2H, Hꢀ1’, J=7.02 Hz), 7.05 (d, 2H, Hꢀ
’’+Hꢀ6’’, J=8.85 Hz), 7.17 (brs, 2H, D O exch., NH ), 7.75 (s, 1H, Hꢀ5’’’), 7.85 (d,
H, Hꢀ3’’+Hꢀ5’’, J=8.85 Hz), 8.14 (s, 1H, Hꢀ2), 8.17 (s, 1H, Hꢀ8). CꢀNMR (50
2
2
13
MHz, DMSOꢀd ) δ 25.68 (Cꢀ3’), 26.12 (Cꢀ2’), 42.57 (Cꢀ1’), 67.44 (Cꢀ4’), 115.48 (Cꢀ
6
2
’’+Cꢀ6’’), 118.75 (Cꢀ5), 123.59 (Cꢀ4’’), 128.99 (Cꢀ3’’+Cꢀ5’’), 134.11 (Cꢀ5’’’),
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140.82 (Cꢀ8), 149.56 (Cꢀ4), 152.38 (Cꢀ2), 155.95 (Cꢀ6), 162.00 (Cꢀ1’’), 173.82 (Cꢀ
+
1
’’’), 214.76 (Cꢀ4’’’). HRꢀMS (ESI) m/z: Calcd for C18
416.0668, found 416.0668. Anal. Calcd for C18 OS
6.85. Found: C, 51.89; H, 4.33; N, 17.07.
18 5 3
H N OS : [M1+H]
=
H
17
N
5
3
: C, 52.03; H, 4.12; N,
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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35
36
37
38
39
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48
49
50
51
52
53
54
55
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57
58
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60
9
-{4-[(4-carbamothioyl)phenyl]butoxy}adenine (11). This compound was
prepared according to the general procedure described above, using 9ꢀ(4ꢀ
hydroxybutyl)adenine and 4ꢀhydroxythiobenzamide Purification was effected by flash
chromatography using a mixture of chloroform / methanol (from 100/5 up to 100/10)
1
as the eluent. Yield: 29%. M.p. 241ꢀ2 °C (THF). HꢀNMR (400 MHz, DMSOꢀd ) δ
6
1
.70 (m, 2H, Hꢀ3’), 1.97 (m, 2H, Hꢀ2’), 4.05 (t, 2H, Hꢀ4’, J=6.42 Hz), 4.21 (t, 2H, Hꢀ
1’, J=7.06 Hz), 6.92 (d, 2H, Hꢀ2’’+Hꢀ6’’, J=8.96 Hz), 7.17 (brs, 2H, D O exch.,
adenineꢀNH ), 7.93 (d, 2H, Hꢀ3’’+Hꢀ5’’, J=8.96 Hz), 8.14 (s, 1H, Hꢀ2), 8.16 (s, 1H,
Hꢀ8), 9.29 (brs, 1H, D O exch., NHꢀthiocarboxamide), 9.61 (brs, 1H, D O exch., NHꢀ
2
2
2
2
13
thiocarboxamide). CꢀNMR (50 MHz, DMSOꢀd ) δ 25.76 (Cꢀ3’), 26.17 (Cꢀ2’), 42.59
6
(Cꢀ1’), 67.19 (Cꢀ4’), 113.47 (Cꢀ2’’+Cꢀ6’’), 118.75 (Cꢀ5), 129.42 (Cꢀ3’’+Cꢀ5’’),
1
1
31.26 (Cꢀ4’’), 140.82 (Cꢀ8), 149.56 (Cꢀ4), 152.37 (Cꢀ2), 155.95 (Cꢀ6), 161.18 (Cꢀ
+
’’), 198.56 (C=S). HRꢀMS (ESI) m/z: Calcd for C16
H
19
N
6
OS: [M1+H] =343.1336,
found 343.1339. Anal. Calcd for C16
6.01; H, 5.22; N, 24.33.
18 6
H N OS: C, 56.12; H, 5.30; N, 24.54. Found: C,
5
9-{4-[(4-carbamoyl)phenyl]butoxy}adenine (12). This compound was
prepared according to the general procedure described above, using 9ꢀ(4ꢀ
hydroxybutyl)adenine and 4ꢀhydroxybenzamide. Purification was effected by flash
chromatography using a mixture of dichloromethane / methanol (from 100/3 up to
1
1
00/10) as the eluent. Yield: 33%. M.p. 267ꢀ8 °C (MeOH). HꢀNMR (400 MHz,
DMSOꢀd
6
) δ 1.69 (m, 2H, Hꢀ3’), 1.97 (m, 2H, Hꢀ2’), 4.04 (t, 2H, Hꢀ4’, J=6.35 Hz),
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4.22 (t, 2H, Hꢀ1’, J=7.00 Hz), 6.94 (d, 2H, Hꢀ2’’+Hꢀ6’’, J=8.77 Hz), 7.14 (brs, 1H,
D
D
2
2
O exch., NHꢀcarboxamide), 7.18 (brs, 2H, D
2 2
O exch., adenineꢀNH ), 7.79 (brs, 1H,
O exch., NHꢀcarboxamide), 7.82 (d, 2H, Hꢀ3’’+Hꢀ5’’, J=8.77 Hz), 8.14 (s, 1H, Hꢀ
13
2
), 8.17 (s, 1H, Hꢀ8). CꢀNMR (50 MHz, DMSOꢀd ) δ 25.77 (Cꢀ3’), 26.18 (Cꢀ2’),
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
42.60 (Cꢀ1’), 67.05 (Cꢀ4’), 113.81 (Cꢀ2’’+Cꢀ6’’), 118.75 (Cꢀ5), 126.44 (Cꢀ4’’),
1
29.29 (Cꢀ3’’+Cꢀ5’’), 140.82 (Cꢀ8), 149.56 (Cꢀ4), 152.37 (Cꢀ2), 155.95 (Cꢀ6), 160.85
+
19 6 2
(Cꢀ1’’), 167.36 (C=O). HRꢀMS (ESI) m/z: Calcd for C16H N O : [M1+H]
=
18 6 2
327.1564, found 327.1568. Anal. Calcd for C16H N O : C, 58.89; H, 5.56; N,
2
5.75. Found: C, 58.71; H, 5.49; N, 25.99.
General procedure for the synthesis of 4 and 6. Trifluoroacetic acid (2 mL)
was added to a solution of compound 3 (0.226 mmol) or 5 (0.194 mmol) in
dichloromethane (3 mL) and the mixture was stirred at room temperature for 20
hours. Upon completion of reaction the solvents were evaporated, the residue was
dissolved in methanol and sodium hydrogen carbonate was added into the flask. The
mixture was stirred at room temperature for 20 minutes, the residue was dryꢀpacked
and the crude product was purified by flash chromatography to provide pure
nucleosides 4 and 6.
5’-O-(4-carbamothioylphenyl)adenosine (4). This compound was prepared
according to the general procedure described above, starting from 3. Purification was
effected by flash chromatography using a mixture of dichloromethane / methanol
1
(from 100/5 up to 100/12) as the eluent. Yield: 86%. M.p. 231ꢀ3 °C (EtOH). HꢀNMR
(
400 MHz, DMSOꢀd
’), 4.72 (m, 1H, Hꢀ2’), 5.41 (d, 1H, D
exch., OHꢀ2’, J=5.85 Hz), 5.97 (d, 1H, Hꢀ1’, J=5.09 Hz), 6.99 (d, 2H, Hꢀ2’’+Hꢀ6’’,
J=8.91 Hz), 7.28 (brs, 2H, D O exch., adenineꢀNH ), 7.94 (d, 2H, Hꢀ3’’+Hꢀ5’’,
6
) δ 4.23ꢀ4.30 (m, 2H, Hꢀ5’+Hꢀ4’), 4.31ꢀ4.36 (m, 2H, Hꢀ5’+Hꢀ
3
2
O exch., OHꢀ3’, J=5.34 Hz), 5.60 (d, 1H, D O
2
2
2
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9
2
J=8.91 Hz), 8.15 (s, 1H, Hꢀ2), 8.33 (s, 1H, Hꢀ8), 9.32 (brs, 1H, D O exch., NHꢀ
13
thiocarboxamide), 9.64 (brs, 1H, D
MHz, DMSOꢀd ) δ 68.10 (Cꢀ5’), 70.41 (Cꢀ3’), 73.12 (Cꢀ2’), 82.14 (Cꢀ4’), 87.64 (Cꢀ
’), 113.54 (Cꢀ2’’+Cꢀ6’’), 119.09 (Cꢀ5), 129.47 (Cꢀ3’’+Cꢀ5’’), 131.63 (Cꢀ4’’), 139.44
2
O exch., NHꢀthiocarboxamide). CꢀNMR (50
6
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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(Cꢀ8), 149.43 (Cꢀ4), 152.69 (Cꢀ2), 156.07 (Cꢀ6), 160.91 (Cꢀ1’’), 198.54 (C=S). HRꢀ
+
MS (ESI) m/z: Calcd for C17
Calcd for C17
0.99.
H
19
N
6
O
4
S: [M1+H] =403.1183, found 403.1183. Anal.
18 6 4
H N O S: C, 50.74; H, 4.51; N, 20.88. Found: C, 50.49; H, 4.66; N,
2
5
’-O-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl]adenosine (6). This compound
was prepared according to the general procedure described above, starting from 5.
Purification was effected by flash chromatography using mixture of
a
dichloromethane / methanol (from 100/5 up to 100/10) as the eluent. Yield: 64%.
1
M.p. 254ꢀ6 °C (MeOH). HꢀNMR (400 MHz, DMSOꢀd
6
) δ 4.26 (m, 1H, Hꢀ4’), 4.31ꢀ
4
.40 (m, 3H, 2xHꢀ5’ + Hꢀ3’), 4.73 (m, 1H, Hꢀ2’), 5.41 (d, 1H, D O exch., OHꢀ3’,
2
J=5.32 Hz), 5.60 (d, 1H, D
2
O exch., OHꢀ2’, J=5.84 Hz), 5.97 (d, 1H, Hꢀ1’, J=5.09
O exch., NH ), 7.77 (s,
H, Hꢀ5’’’), 7.88 (d, 2H, Hꢀ3’’+Hꢀ5’’, J=8.86 Hz), 8.15 (s, 1H, Hꢀ2), 8.33 (s, 1H, Hꢀ
Hz), 7.12 (d, 2H, Hꢀ2’’+Hꢀ6’’, J=8.86 Hz), 7.28 (brs, 2H, D
2
2
1
8
13
6
). CꢀNMR (50 MHz, DMSOꢀd ) δ 68.26 (Cꢀ5’), 70.36 (Cꢀ3’), 73.06 (Cꢀ2’), 82.05
(Cꢀ4’), 87.66 (Cꢀ1’), 115.54 (Cꢀ2’’+Cꢀ6’’), 119.09 (Cꢀ5), 123.94 (Cꢀ4’’), 129.04 (Cꢀ
3’’+Cꢀ5’’), 134.26 (Cꢀ5’’’), 139.48 (Cꢀ8), 149.40 (Cꢀ4), 152.66 (Cꢀ2), 156.05 (Cꢀ6),
1
61.72 (Cꢀ1’’), 173.71 (Cꢀ1’’’), 214.83 (Cꢀ4’’’). HRꢀMS (ESI) m/z: Calcd for
+
C
19
H
18
N
5
O
4
S
3
: [M1+H] =476.0515, found 476.0513. Anal. Calcd for C19
H
17
N
5
O
4
3
S :
C, 47.99; H, 3.60; N, 14.73. Found: C, 48.22; H, 3.81; N, 14.84.
9-(4-Acetoxybutyl)adenine (8). 9ꢀ(4ꢀAcetoxybutyl)adenine was prepared
30
13
according to a previously described method. M.p. 177ꢀ8 °C (ref. 176ꢀ8 °C). Cꢀ
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4
4
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4
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5
5
5
5
5
5
6
6
NMR data, not provided in previous publication: (50 MHz, DMSOꢀd ) δ 20.65
(CH3CO), 25.32 (Cꢀ3’), 26.07 (Cꢀ2’), 42.52 (Cꢀ1’), 63.24 (Cꢀ4’), 118.77 (Cꢀ5),
1
40.86 (Cꢀ8), 149.58 (Cꢀ4), 152.42 (Cꢀ2), 155.99 (Cꢀ6), 170.38 (COCH3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
9-(4-Hydroxybutyl)adenine (9). This was prepared according to a previously
30
described method with the modification that the deprotection was performed at
1
3
room temperature in quantitative yield. M.p. 200ꢀ2 °C (ref. 200ꢀ2 °C). CꢀNMR data,
not provided in previous publication: (50 MHz, DMSOꢀd ) δ 26.33 (Cꢀ2’), 29.52 (Cꢀ
’), 42.91 (Cꢀ1’), 60.18 (Cꢀ4’), 118.79 (Cꢀ5), 140.93 (Cꢀ8), 149.60 (Cꢀ4), 152.40 (Cꢀ
), 155.98 (Cꢀ6).
6
3
2
In vitro hydrogen sulfide release evaluation
2 2
H S determination (methylene blue assay). H S determination was
43
performed according to Stipanuk and Beck (1982), with some modifications.
Compounds were added to a reaction mixture (total volume 500ꢁL) containing
potassium phosphate buffer (100 mM, pH 7.4) in a sealed microcentrifuge tube and
incubated at room temperature for 15 min. 250ꢁL of Zinc acetate (10%) were then
added to trap H
133ꢁL of N,Nꢀdimethylꢀpꢀphenylenediamine sulphate (DPD, 20 mM, in 7.2 M HCl)
and 133ꢁL of FeCl (30mM, in 1.2 M HCl) were added. Absorbance was measured at
50 nm after 20min. All samples were assayed in triplicate.
2
S followed by 250ꢁL of tricloracetic acid (TCA, 10%). Subsequently,
3
6
Amperometric H S detection. Kinetics of H S release was followed
2
2
amperometrically using a selective H
2
S electrode connected to Free Radical Analyzer
2
(World Precision Instruments). Electrode was calibrated using standard Na S
44
solutions as described previously.
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9
Ultra-high resolution mass spectrometry. Mass spectrometry was performed
on maXis 5G, Bruker Daltonik (Bremen, Germany), an ESIꢀToF MS capable of
resolution of at least 40.000 FWHM. Detection was in positiveꢀion mode. For the
realꢀtime measurements, concentrated DMSO stock solutions of each compounds
were diluted fast with 20 mM ammonium carbonate buffer pH 7.4 (final concentration
of the compound 100 ꢁM) in the injection syringe (10 s) and then continuously
injected into maXis (180 ꢁL/h). Spectra were recorded over 40 min time.
10
11
12
13
14
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17
18
19
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In vitro evaluation of cGMP measurements. Confluent rat lung fibroblasts
(RLFꢀ6) were washed with phosphate buffered saline and incubated in Hanks’
balanced salt solution (HBSS) with vehicle (basal), Na S or the indicated
2
concentration of the compounds tested for 5 min. After the treatment, cells were
washed with Hanks’ balanced salt solution and cGMP was extracted using 0.1 N HCl.
cGMP content was measured in the extracts using a commercially available enzyme
immunoassay kit following the manufacturer’s instructions (Enzo Life Sciences).
In vivo evaluation of the cardioprotective activity of the tested compounds
Surgical preparation. All animal procedures were in compliance with the
European Community guidelines for the use of experimental animals; experimental
protocols were approved by the Ethical Committee of the Prefecture of Athens.
Animals received standard laboratory diet. The initial animal cohort compromised of
88 New Zealand white male rabbits.
.
ꢀ1
Sodium thiopentone at a dose of 30 mg kg was injected into a peripheral vein
for anesthesia. Then the animals were intubated and connected to a respirator for
small animals (MD Industries, Mobile, AL, USA) for mechanical ventilation at a rate
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2
2
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3
3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
adjusted to maintain normal blood gases. Two catheters were inserted; one in the
jugular vein for fluids and anesthetic and one in the carotid artery for blood pressure
monitoring via a transducer attached to a multichannel recorder (Power lab 4.0,
Adinstruments, UK). The chest was surgically opened with a left thoracotomy and the
beating heart was exposed. The pericardium was opened and a 3ꢀ0 silk suture was
passed around a prominent coronary artery. Ischemia was induced by pulling the
thread through a small piece of soft tubing, which was firmly positioned against the
coronary arterial wall with the aid of a small clamp. Ischemia resulted in ST elevation
on the ECG (Power lab 4.0, Adinstruments, UK). At the end of ischemic period the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
45
snare was opened, the artery refilled and the myocardium reperfused.
Experimental protocol. New Zealand white male rabbits weighing 2.8ꢀ3.1 kg
were subjected to 30 minutes regional ischemia of the myocardium, followed by 3
hours of reperfusion and were randomized into 13 groups as follows (experimental
protocol presented in detail in Figure 5):
1
2
) Control group (n=6), no further intervention
) PostC group (n=6): Application of 8 cycles of 30 sec ischemiaꢀreperfusion
immediately after sustained ischemia.
.
ꢀ1
3
) 11 group (n=6): treated with compound 11 at a dose of 1.79 ꢁmol kg bolus on the
th
2
0 min of isc;
.
ꢀ1
4
) 11dd group (n=5): treated with compound 11 at a dose of 3.58 ꢁmol kg bolus on
th
the 20 min of isc;
.
ꢀ1
5) 4 group (n=6): treated with compound 4, at a dose of 1.79 ꢁmol kg bolus on the
th
2
0 min of isc;
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9
6) 9 group (n=6): treated with the compound 9ꢀ(4ꢀhydroxybutyl)adenine (9) at a dose
.
ꢀ1
th
of 1.79 ꢁmol kg at the 20 min of sustained ischemia;
.
ꢀ1
7
2
) 12 group (n=6): treated with compound 12, at a dose of 1.79 ꢁmol kg bolus on the
10
11
12
13
14
15
16
17
18
19
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60
th
0 min of isc;
8
) 4-OH TBZ group (n=6): treated with compound 4-OH TBZ, at a dose of 1.79
.
ꢀ1
th
ꢁ
mol kg bolus on the 20 min of isc;
.
ꢀ1
.
ꢀ1
9
) NaHS group (n=5): treated with NaHS at a dose of 100 ꢁg kg (1.79 ꢁmol kg )
th
.
ꢀ1. ꢀ1
ꢀ1. ꢀ1
bolus on the 20 min of isc followed by infusion of 1 mg kg h (17.9 ꢁmol kg h )
12
for the next 120 min as we have previously described.
1
0) 11+SPT group (n=5): treated with 11 and the adenosine receptor blocker 8ꢀ(p
.
ꢀ1
sulfophenyl)theophylline (SPT), at a dose of 10 mg kg 20 min before sustained
46
ischemia as we have previously described.
46
1
1
1
1) 4+SPT group (n=5): treated with 4 and SPT as previously described.
46
2) 12+SPT group (n=5): treated with 12 and SPT as previously described.
4
6
3) 12+NaHS group (n=5): treated with 12 and NaHS as previously described.
Doses of 4 and 11 were estimated at 20ꢀfold lower dose compared to NaHS.
Heart rate and blood pressure were measured immediately before sustained ischemia
th
(baseline), at the 20 minute of sustained ischemia (one minute after the
administration of the compounds) and at the end of long reperfusion. Blood samples
th
from all groups were collected at the 20 min of reperfusion for the determination of
thiosulfates.
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5
6
In a second series of experiments, rabbits (4 per group of Control, NaHS, 4
th
and 11 groups) were subjected to the same interventions up to the 10 minute of
reperfusion, when tissue samples from the ischemic area of myocardium were
collected, snapꢀfrozen in liquid nitrogen and stored at ꢀ80°C for western blot analysis
of vasodilatorꢀstimulated phosphoprotein (VASP), and phospholamban (PLN).
0
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9
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1
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3
4
5
6
7
8
9
0
Risk area and infract size. At the end of reperfusion time rabbits were
sacrificed with a high dose intravenous injection of the anesthetic sodium thiopentone
.
ꢀ1
(150 mg kg ), hearts were rapidly excised, mounted on an apparatus and perfused
with normal saline for 2 minutes for blood removal. Then the coronary ligature was
retightened at the same site and 10 mL of green fluorescent microspheres (Duke
Scientific Corp. Palo Alto CA) were infused for the separation of the normally
perfused area from the area at risk. Hearts were kept in the refrigerator for 24 hours
and cut into 3 mm thick sections. The slices were stained with triphenyltetrazolium
chloride (TTC) at 37°C and immersed in formaldehyde. With a wavelength of 366 nm
UV light, we identified the risk zone and separated it from the infarcted zone of the
heart. The infarcted, the risk and the normal areas were traced onto an acetate sheet,
which had been placed over the top glass plate. The tracings were subsequently
scanned with the Abode Photoshop 6.0 and measured with the Scion Image program.
The areas of myocardial tissue at risk and infarcted were automatically transformed
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into volumes. Infarct and risk area volumes were expressed in cm and the percent of
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infarct to risk area ratio (%I/R) was calculated.
Western Blot Analysis. Tissues from the ischemic area of myocardium were
pulverized and the sample powder was homogenized in lysis solution (1% Triton
X100, 20 mM Tris pH 7.4ꢀ7.6, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM
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EGTA, 1 mM Glycerolphosphatase, 1% SDS, 100 mM PMSF, 0.1% Protease
phosphatase inhibitor cocktail) and homogenized. After centrifugation at 11,000 g for
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5 min at 4 °C, supernatants were collected. Supernatants were treated with protein G
beads for rabbit IgG removal, in order to minimize the nonspecific binding of the
primary antibody. In brief 40ꢁL of supernatants were incubated for 4 hours with
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00ꢁL of protein G beads. After centrifuging and aspiration of the supernatants, the
samples were reꢀdissolved with lysis buffer and the protein concentration was
measured. The protein concentration was determined based on the Bradford dyeꢀ
binding procedure. The supernatant was mixed with Dave’s buffer (4% SDS, 10% 2ꢀ
mercaptoethanol, 20% glycerol, 0.004% bromophenyl blue, 0.125 M Tris/HCl). The
samples were heated at 100 °C for 10 min and stored at ꢀ80 °C. An equal amount of
protein was loaded into each well and then separated by sodium dodecylsulfateꢀ
polyacrylamide gel electrophoresis 7.5ꢀ11% and transferred onto a polyvinylidene
difluoride membrane. After blocking with 5% nonꢀfat dry milk, membranes were
incubated overnight at 4 °C with the following primary antibodies: phospho VASP
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Ser239), phospho PLN (ser16, tyr17), and betaꢀactin (Cell Signaling Technology,
Beverly, MA, USA). Membranes were then incubated with secondary antibodies for
–2 h at room temperature (goat antiꢀmouse and goat antiꢀrabbit HRP, Biorad,
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Interlab, Athens, Greece) and developed using the GE Healthcare ECL Western
Blotting Detection Reagents (Thermo Scientific Technologies, Bioanalytica, Athens,
Greece). Relative densitometry was determined using a computerized software
package (NIH Image, National Institutes of Health, USA) and the values for
phosphorylated VASP and PLN (ser16/thr17) were normalized to the values for betaꢀ
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actin.
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Data analysis and statistics. All results are presented as means + standard
error of mean (SEM). Data of in vitro and in vivo experiments were compared by oneꢀ
way analysis of variance (ANOVA) with Bonferroni correction and Tukey postꢀhoc
analysis. A calculated P value of less than 0.05 was considered to be statistically
significant.
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Determination of plasma levels of thiosulfates with the UPLC-DAD method
Instrumentation. All chromatographic analyses were performed using a
Waters Acquity UPLC system comprising of a Solvent Manager Module connected to
a Sample Manager Module. Detection has been performed using an Acquity PDA
Detector (Waters Corporation, Manchester, UK). For the separation of the analyte,
UKtes a Fortis C18 column (50×2.1 mm, 1.7 ꢁm, Cheshire, UK) has been employed.
For the centrifugation procedures a Hettich Universal 32R (Buckinghamshire, UK)
instrument has been used, whereas solvent evaporation was performed using a
Thermo Scientific ReactiꢀVap apparatus.
Chemicals. All solvents used throughout the analytical experiments were
purchased from Merck and were of HPLC grade of purity. Anhydrous monopotassium
phosphate and phosphoric acid have also been obtained from Merck whereas sodium
thiosulfate and aꢀnaphthol were obtained from SigmaꢀAldrich. CMQT has been
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synthesized in our laboratory using the method of literature and its purity has been
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evaluated by HꢀNMR and was greater than 95%.
Sample preparation. To 100 ꢁL plasma an ice cold 200 ꢁL aliquot of a
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solution of the ISTD in ACN (38.57 ꢁmol L ) has been added, the mixture was
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vortexed for 1 min and centrifuged to 16000 rpm for 15 min at 4 °C. Subsequently
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70 ꢁL of the supernatant were transferred to screw capped 1.5 mL vials and 10 ꢁL of
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CMQT (0.1 mol L ) were added along with 260 ꢁL of phosphate buffer (0.1 mol L ,
pH=2). The mixture remained for 5 min at room temperature and 5 ꢁL were injected
to the UPLC.
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Chromatographic separation. A gradient elution program has been
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developed for the separation of the analyte the ISTD using H O as solvent A and
ACN as solvent B. Briefly a linear gradient has been employed from 20% to 100% B
in 3 min and the column was eluted for an additional 0.2 min at 100% B. The flow
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rate was kept at 0.6 mL min whereas the column temperature was maintained at 40
°C. For the detection of the eluting substances a λmax of 375 nm has been used for
MTQ and 254 nm for the ISTD. Under these conditions MTQ was eluting at a t
.2 min and ISTD at 1.35 min with a USP calculated resolution of 4.4.
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Internal standard choice. Initially thiophenol has been employed as the ISTD
but severe stability and slow reaction problems rendered its use impractical whereas
the ethanedithiol used resulted in extended dimerization. Thus a nonꢀreactive ISTD
has been finally employed in order to test the recovery and the instrumental
performance but with the deficiency of not checking for the reaction.
Validation. The methodology has been validated using rabbit samples, spiked
with thiosulfate. In order to evaluate the specificity of the method 10 plasma samples
from different rabbits have been used. Linearity has been examined constructing a
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calibration curve in the dynamic range of 0.25ꢀ20 ꢁmol L using 7 spiked plasma
samples whereas the repeatability (n=5) and the intermediate precision (for three
consecutive days) were tested using 5 QC samples namely QC =LLOQ,
L
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