Synthesis and Pharmacological Evaluation of Novel Adenine

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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, AlexiosLeandros 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

SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF NOVEL ADENINE-HYDROGEN SULFIDE SLOW RELEASE HYBRIDS DESIGNED AS MULTI-TARGET CARDIOPROTECTIVE AGENTS Nikolaos Lougiakis,a Andreas Papapetropoulos,a Evangelos Gikas,a Spyridon Toumpas,a Panagiotis Efentakis,a Rudolf Wedmann,b Anastasia Zoga,a,c Zhongmin Zhou,a Efstathios K. Iliodromitis,c Alexios-Leandros Skaltsounis,d Milos R. Filipovic,b Nicole Pouli,a* Panagiotis Marakos,a Ioanna Andreadoua* a

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, National and

Kapodistrian University of Athens, Greece b

Department of Chemistry and Pharmacy, Friedrich-Alexander University of

Erlangen-Nuremberg, 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 7274827; fax:+30 210 7274747; e-mail: [email protected] Nicole Pouli, National and Kapodistrian University of Athens, Faculty of Pharmacy, Panepistimiopolis Zografou, Athens 15771, GREECE; tel:+30 210 7274185; fax:+30 210 7274747; e-mail: [email protected]

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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 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.


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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 re-establish the coronary blood flow, resulting in a significant decrease of the ischemia-related damage. However, reperfusion itself contributes to the total injury and causes a number of deleterious effects to the ischemic myocardial tissue, a procedure known as reperfusion injury.1 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 injury.2 Although PostC has been shown to be successful in preclinical models, it has failed to translate into therapeutically useful cardioprotective strategies that impact everyday clinical practice.3 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 for protection against lethal reperfusion injury.4 To achieve this goal different pharmacological manipulations of the cellular events have been proposed based on the advances in our understanding of the involved pathogenetic mechanisms.5,6 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 inhibition of the mitochondrial permeability transition pore (mPTP) opening.7 mPTP opening, which plays a key role in myocardial necrosis at reperfusion,8 is regulated by several factors, including Ca2+ overload, mitochondrial matrix pH, decrease of



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membrane potential, and excessive production of reactive oxygen species (ROS).9 Thus, selective inhibition of mPTP opening appears to be a promising therapeutic strategy against myocardial ischemia-reperfusion injuries. Although a number of studies have demonstrated the cardioprotective effects of endogenous H2S in myocardial ischemia/reperfusion injury,10,11 there exist limited reports concerning the cardioprotective effects of exogenous administration of H2S in models of ischemia/reperfusion.10 We have recently shown that administration of sodium hydrogen sulfide (NaHS, a salt of H2S) induces cardioprotection via a cGMP/PKG/phosholamban (PLN) pathway thereby preventing mPTP opening.12 However, the exposure of biological systems to inorganic H2S salts, causes a burst of H2S that does not recapitulate the low level of continuous production of H2S that occurs in vivo. For these reasons, the discovery of pharmacologically useful precursors for endogenous H2S synthesis or, alternatively the use of H2S-slow releasing compounds seems to be a convenient and compelling strategy.13,14 An established medicinal chemistry approach in this field deals with the covalent incorporation of a H2S donating group into the structure of a biologically active derivative. Thus, a number of H2S releasing moieties have been developed, including morpholin-4-ium

4-methoxyphenylmorpholinophosphinodithioate

(GYY4137,

a

water soluble derivative of the Lawesson’s reagent), arylthiobenzamides, 4hydroxythiobenzamide (4-OH-TBZ), anethole dithiolethione

(ADT) and its

metabolite 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH).15,16 A number of hybrid compounds are currently under intensive investigation possessing a broad spectrum of activities,17 and in particular some H2S-releasing nonsteroidal antiinflammatory drug (NSAID) derivatives have been reported to display increased effectiveness and reduced toxicity, when compared to their parent drugs.14, 15, 18


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However, there are limited novel H2S releasing molecules in the literature, acting either as slow,16 or controllable releasers,19 with protective effects against ischemia/ reperfusion injury. To the best of our knowledge there are no reports of hybrids possessing H2S-releasing properties that when administered at a clinical 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 ischemia/reperfusion injury.20 Exogenously administered adenosine and its selective agonists reduced infarct size, but this benefit was not translated into improved clinical outcome.21,22 Additionally, the ability of intracoronary adenosine infusion immediately before coronary stenting to limit infarct size has been tested in clinical trials with disappointing results.23,24 These controversial data might be attributed to the use of inadequate doses of the drug, which are not high enough to overcome adenosine’s extremely rapid metabolism in red blood cells and endothelial cells.25 Furthermore, the high dose required to exert cardioprotective effects, can be associated with severe hypotension and bradycardia, due to vasodilatory and negative chronotropic effects of the drug.25,26 These hemodynamic consequences limit the usefulness of adenosine, as a cardioprotective agent in humans.26 Having in mind the beneficial effects of both adenosine and hydrogen sulfide in the cardiovascular system during ischemia/reperfusion, we hypothesized that H2Sreleasing 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 non-nucleoside analogue of adenine, 9(4-hydroxybutyl)adenine, were coupled with 4-OH-TBZ and ADT-OH (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. In order to assess the effectiveness of these compounds in releasing H2S in vivo, a UPLC-DAD 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 H2S concentrations 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 thiosulfate and sulfate by specific enzymes.27 Since most of the sulfate is derived from H2S-independent sources, such as cysteine oxidation, thiosulfate is considered the most specific marker of H2S metabolism.28

RESULTS AND DISCUSSION Chemistry. 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH) 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

5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione,

which was

finally

demethylated upon heating with pyridine HCl.29 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 ADTOH or commercially available 4-hydroxythiobenzamide, in the presence of DIAD and


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PPh3. 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 9-(4-hydroxybutyl)acetate 8 upon treatment with 4bromobutylacetate under basic conditions and the isolated N-9 isomer was subsequently treated with methanolic ammonia at room temperature, providing the alcohol 9.30 Mitsunobu type reaction of 9 with ADT-OH, 4-OH-TBZ, or 4hydroxybenzamide, resulted into the target compounds 10-12. In vitro hydrogen sulfide release evaluation. The newly synthesized compounds 4, 6, 10 and 11, together with the intermediate isopropylidene-protected 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 reported one to measure hydrogen sulfide in biological samples.31,32 It is based on the colorimetric determination of methylene blue dye, resulting from the reaction of sulfide with N,N-dimethyl-p-phenylenediamine sulfate in the presence of Fe3+ as an oxidizing agent. In this assay all compounds demonstrated a concentration-dependent H2S release. Nevertheless, the 4-OH-TBZ derivatives 3, 4 and 11 produced high amounts of H2S, whereas the isopropylidene derivative 5 as well as the 5’-adenosine and adenine ethers 6 and 10 respectively, all bearing the ADT-OH substitution, released lower levels of H2S (Figure 2A). To further confirm the H2S-donating ability of these compounds we used amperometric method with selective H2S electrode. All compounds showed slow and steady H2S release. The representative electrode trace of one of the compounds (compound 4) is shown in Figure 2. Compound 4 showed biphasic H2S release: an instantaneous release of small H2S amounts was noticeable when DMSO stock



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solution was added to buffer, followed by very slow and steady H2S release (Figure 2B). Control electrode trace of 10 µM Na2S showed completely opposite kinetics: a constant decay due to the fact that the whole amount of sulfide ended up as H2S, which then slowly oxidized and got physically removed from the system (Figure 2C). Addition of compound 12, which has no H2S donating properties, led to no change in electrode response (Figure 2C). Interestingly, addition of 1 mM glutathione (GSH) slowed down the H2S release from compound 4, suggesting an interaction between the two molecules (Figure 2D). We should mention that the results obtained with the H2S electrode and the methylene blue method are qualitatively similar. In both cases, when a low concentration of compound 4 was used, no H2S could be detected, while the higher concentration (150 µM for the methylene blue and 100 µM for the electrode measurements) yielded measurable H2S amounts. Characterization of H2S release mechanism. To elucidate the possible mechanism of H2S release by this class of compounds, we used time-resolved UHRESI-TOF-MS. 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 [C17H18N6O5+H]+ (calculated m/z 387.1411) (Figure 3B), as well as of m/z 371.1464 peak which corresponds to the [C17H18N6O4+H]+ (calculated m/z 371.1462) were immediately noticeable (Figure 3C). This suggests that possible mechanism for H2S 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 H2S release from 100 µM of compound 4 to be 0.8±0.1 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 could be observed at m/z 340.0636 (calculated m/z 340.0632) (Figure 3F), and at m/z 647.1475 corresponding to the cluster of [GSH + GSSH + H]+ (calculated 647.1470, not shown). This suggests that under physiological conditions, in parallel to a direct H2S release this class of compounds could lead to the formation of low molecular weight persulfides, which could explain the slower H2S release observed in Figure 2D. GSSH has been recently suggested to be one of the main mediators of H2S biological effects leading to trans-persulfidation of protein targets.33,34 Considering mM concentration of GSH in the cells, it is tempting to speculate that biological effects of this type of H2S donors will partly, if not largely, derive from the formation of GSSH and subsequent protein persulfidation. In vitro evaluation of cGMP measurements. As we have previously shown, H2S increases cGMP by inhibiting phosphodiesterase (PDE) and by enhancing eNOS activity.35,36 Many studies have also showed a consistent protective effect of interventions aimed to increase cGMP in reperfused myocardium.37-39 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 that yielded more H2S in the methylene blue assay, namely 3, 4 and 11, also caused a substantial increase in cGMP (Figure 4). It should be noted that 9-(4hydroxybutyl)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 H2S-releasing moiety (4-OH-TBZ), suggesting that the adenine nucleus contributes in the stabilization of the molecule in the cellular milieu and



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reduces the rate of H2S generation. Based on these results and taking under consideration that compounds that could release an adequate amount of H2S may possibly have a better pharmacological effect, compounds 4 and 11, which clearly possess the better profile as H2S donors, were chosen for further evaluation to the in vivo experiments. In vivo evaluation of the cardioprotective activity of the tested compounds. Recent studies with animal models suggested that H2S can protect cardiovascular system against myocardial ischemia/reperfusion injury.10,16,19 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 corresponding dose of NaHS12 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

Synthesis and Pharmacological Evaluation of Novel Adenine

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