Trends in H2S-Donors Chemistry and Their Effects in Cardiovascular Diseases

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Review Trends in H2S-Donors Chemistry and Their Effects in Cardiovascular Diseases

Angela Corvino , Francesco Frecentese , Elisa Magli , Elisa Perissutti , Vincenzo Santagada, Antonia Scognamiglio , Giuseppe Caliendo, Ferdinando Fiorino and Beatrice Severino *

Department of Pharmacy, School of Medicine, University of Naples Federico II, Via D. Montesano, 49, 80131 Napoli, Italy; [email protected] (A.C.); [email protected] (F.F.); [email protected] (E.M.); [email protected] (E.P.); [email protected] (V.S.); [email protected] (A.S.); [email protected] (G.C.); feﬁ[email protected] (F.F.) * Correspondence: [email protected]; Tel.: +39-081-679-828

Abstract: Hydrogen sulfide (H2S) is an endogenous gasotransmitter recently emerged as an important regulatory mediator of numerous human cell functions in health and in disease. In fact, much evidence has suggested that hydrogen sulfide plays a significant role in many physio-pathological processes, such as inflammation, oxidation, neurophysiology, ion channels regulation, cardiovascular protection, endocrine regulation, and tumor progression. Considering the plethora of physiological

effects of this gasotransmitter, the protective role of H2S donors in different disease models has been extensively studied. Based on the growing interest in H2S-releasing compounds and their importance as tools for biological and pharmacological studies, this review is an exploration of currently available

H2S donors, classifying them by the H2S-releasing-triggered mechanism and highlighting those potentially useful as promising drugs in the treatment of cardiovascular diseases.

Citation: Corvino, A.; Frecentese, F.; Magli, E.; Perissutti, E.; Santagada, V.; Keywords: hydrogen sulﬁde; natural H2S donors; synthetic H2S donors; triggered mechanism; H2S Scognamiglio, A.; Caliendo, G.; release; cardiovascular diseases Fiorino, F.; Severino, B. Trends in

H2S-Donors Chemistry and Their Effects in Cardiovascular Diseases. Antioxidants 2021, 10, 429. https:// 1. Introduction doi.org/10.3390/antiox10030429 Hydrogen sulfide (H2S), the third endogenous recognized gaseous signaling transmitter [1], among nitric oxide (NO) and carbon monoxide (CO), is a colorless and pungent gas Academic Editor: Daniele Mancard with a boiling point of 60 ◦C[2]. It is endogenously synthesized by four enzymes: cystathionine γ-lyase (CSE) and cys- Received: 17 February 2021 tathionine β-synthetase (CBS), which catalyze the production of H2S by a direct enzymatic Accepted: 8 March 2021 desulfhydration of L-cysteine, and 3-mercaptopyruvate sulfurtransferase (3-MST), which Published: 11 March 2021 produces H2S by an indirect desulfhydration, in concert with cysteine aminotransferase (CAT) and in the presence of reductants [3]. The expression of these enzymes is tissue Publisher’s Note: MDPI stays neutral specific. The amount of CBS is found mostly in the central nervous system, the liver, with regard to jurisdictional claims in the kidney, the uterus, and placenta; CSE is primarily concentrated in the cardiovascular published maps and institutional affil- system, whereas 3-MST is located predominantly in the mitochondria [4–8]. iations. To study the biological effects of endogenously synthesized H2S, the development of inhibitors selective for the above enzymes is required. To date, some selective inhibitors of CSE and CBS have been identified and widely used in biological systems [9–13]; conversely, no pharmacological inhibitors of the enzyme 3-MST have yet been discovered. Copyright: © 2021 by the authors. Several studies have shown that H2S plays a significant modulatory role in numer- Licensee MDPI, Basel, Switzerland. ous physio-pathological processes in the human body [14]. In fact, it is involved in the This article is an open access article homeostatic regulation of respiratory, cardiovascular, immune, nervous, gastroenteric, and distributed under the terms and endocrine systems [4,15]. conditions of the Creative Commons Currently, growing research evidence has confirmed the protective effects of H S in Attribution (CC BY) license (https:// 2 creativecommons.org/licenses/by/ cardiovascular diseases [16–23], such as cardiac hypertrophy, heart failure, myocardial 4.0/). ischemia/reperfusion (I/R) injury [24], hypertension [18,25] and atherosclerosis [26], acting

Antioxidants 2021, 10, 429. https://doi.org/10.3390/antiox10030429 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, x FOR PEER REVIEW 2 of 27

Antioxidants 2021 10 , , 429 Currently, growing research evidence has confirmed the protective effects of H2 of2S 27 in cardiovascular diseases [16–23], such as cardiac hypertrophy, heart failure, myocardial ischemia/reperfusion (I/R) injury [24], hypertension [18,25] and atherosclerosis [26], acting asas an an activator activator of of angiogenesis angiogenesis [27 ],[27], basal basal vasorelaxant vasorelaxant agent agent [28], blood[28], blood pressure, pressure, and heart and rateheart regulator rate regulator [29,30]. [29,30]. Moreover, Moreover, it has beenit has demonstrated been demonstrated that the that related the related mechanisms mecha- ofnisms action of accountingaction accounting for this for cardioprotective this cardioprotective activity activity involve involve antioxidation, antioxidation, inhibition inhibi- of celltion apoptosis, of cell apoptosis, pro-angiogenesis, pro-angiogenesis, anti-inﬂammatory, anti-inflammatory, and ion channelsand ion channels regulation regulation [31,32] (Figure[31,32]1 (Figure). 1).

Figure 1. Schematic illustration of the effects of H2S in different heart diseases and the molecular Figure 1. Schematic illustration of the effects of H2S in different heart diseases and the molecular mechanisms underlying H2S-induced cardioprotection. mechanisms underlying H2S-induced cardioprotection.

AsAs depicted depicted in in Figure Figure1 ,H1, H2S2S exerts exerts a a cardioprotective cardioprotective effect effect by by activating activating different different endothelium-dependentendothelium-dependent signaling signaling pathways pathways [ 33[33]].. It It reduces reduces blood blood pressure, pressure, by by inducing inducing + vasorelaxation,vasorelaxation, aa consequenceconsequence of opening K KATPATP channelschannels and and increasing increasing K+ K currentscurrents result- re- sultinging in inhyperpolarizing hyperpolarizing membrane membrane of ofsmooth smooth muscle muscle cells cells [34]. [34 ].Moreover, Moreover, H H2S2 showsS shows its itsanti-hypertensive anti-hypertensive effect, effect, by by activating activating endothelial endothelial nitric nitric oxide oxide synthase synthase (eNOS) (eNOS) and and in- increasingcreasing NO NO bioavailability bioavailability [35]. [35]. It It also also shows shows an an inhibitory inhibitory effect effect on on the the pathogenesis pathogenesis of ofatherosclerosis, atherosclerosis, by by preventing preventing an an inflammatory inﬂammatory response response mediated mediated by byinflammatory inﬂammatory cy- cytokinestokines [26] [26 ]and and exerting exerting antioxidative antioxidative action action through thethe activationactivation ofof thethe nuclear nuclear factor factor erythroiderythroid 2-related2-related factorfactor 2 2 (Nrf2)-dependent (Nrf2)-dependent pathwaypathway andand thethe protection protection of of tissues tissues by by reactivereactiveoxygen oxygen speciesspecies (ROS).(ROS). The cardioprotection cardioprotection of of H H2S2S is is also also associated associated with with the the in- inhibitionhibition of of cardiomyocyte cardiomyocyte apoptosis apoptosis after after myoc myocardialardial injury. injury. In Infact, fact, it suppresses it suppresses the the ac- activationtivation of of caspase-3 caspase-3 and and upregulates upregulates the the expr expressionession of glycogen synthasesynthase kinase-3kinase-3 (GSK-(GSK- 33β𝛽).). The The antioxidant antioxidant effect effect of of H H2S2S is is also also embodied embodied in in the the preservation preservation of of mitochondrial mitochondrial functionsfunctions by by inhibiting inhibiting mitochondrial mitochondrial respiration respiration [ 36[36].]. Furthermore, H S stimulates angiogenesis by increasing the expression of VEGF and Furthermore, H2 2S stimulates angiogenesis by increasing the expression of VEGF and activatingactivating some some downstream downstream effectors, effectors, such such as as Akt, Akt, STAT3, STAT3, ERK, ERK, and and p38 p38 [37 [37].]. Recently, considering the interesting involvement of H S in the cardiovascular sys- Recently, considering the interesting involvement of H2 2S in the cardiovascular system,tem, there there has has been been heightened heightened enthusiasm enthusiasm for for the the development development of of compounds compounds able able to to generate exogenous H2S, which could represent biological tools and promising cardiopro- generate exogenous H2S, which could represent biological tools and promising cardioprotective agents. tective agents.

2. H2S Donors 2. H2S Donors For the past several years, inorganic sulfide salts, such as sodium sulfide (Na2S) and hydrosulfideFor the (NaHS),past several have years, been commonlyinorganic sul usedfide as salts, pharmacological such as sodium tools. sulfide They (Na represent2S) and hydrosulfide (NaHS), have been commonly used as pharmacological tools. They the early H2S donors used in biomedicine field, the results of which make it very useful to elucidate the physio-pathological roles of H2S in mammalian systems. Although these sulfide salts have been the basic tools used for H2S research for years, some of their significant limitations were demonstrated. In fact, sulfide salts hydrolyze Antioxidants 2021, 10, 429 3 of 27

immediately upon reaction with water and the resulting too-rapid release of H2S causes its blood and tissue concentrations to surge to supraphysiological levels followed by a rapid drop [38]. Therefore, this suboptimal pharmacokinetic profile led the researchers to limit the use of these salts as potential therapeutics and to obtain novel organic H2S-releasing compounds, including chemically synthesized molecules and natural plant extracts. One of the first slow-releasing H2S donors was Lawesson’s reagent, a synthetic compound which is largely used for sulfurization of organic molecules [39]. It has been used in some pharmacological studies as an H2S donor, despite its lack of water solubility. Therefore, to improve this chemical property, GYY4137, a water-soluble derivative of Lawesson’s reagent, was obtained. Similarly to its parent compound, it also releases H2S upon hydrolysis, but it better mimics the physiological H2S production, due to its slow-releasing nature [40,41]. Moreover, in order to optimize the H2S release properties of GYY4137, structural modifications were made to this compound via substitution of the P-C bond in GYY4137 with a P-O bond, affording a series of O-substituted phosphorodithioate- based H2S donors [42]. Later on, naturally occurring H2S donors derived from some vegetables belonging to Alliaceae and Brassicaceae, such as garlic, onion, broccoli, cabbage, watercress, and garden cress, were also investigated. So far, among garlic-derived compounds, allicin is one of the best characterized. Due to its instability in aqueous media, it quickly decomposes into four H2S-releasing compounds: diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), and ajoene [43]. Other garlic-derived compounds containing a sulfur atom are S-allylcysteine (SAC) with its structural analogs, S-propyl-L-cysteine (SPC), and S-propargyl-cysteine (SPRC), which are potential sources of H2S[44–46]. Similarly, some cruciferous vegetables contain natural isothiocyanates, such as sulforaphane, allyl isothiocyanate, benzyl isothiocyanate, 4-hydroxybenzyl isothiocyanate, and erucin, which exhibit a significant H2S-releasing activity [47]. Moreover, it has been demonstrated that some thioamino acids, such as thioglycine and thiovaline, are also H2S donors, showing a slow release of the gasotransmitter [48]. Recently, the limited clinical application of some naturally occurring H2S donors led to the development of novel synthetic compounds with favorable pharmacological properties, which could be useful both as research tools and as potential therapeutics. Thus, in order to obtain effective H2S donors with controllable release rates, a series of N-(benzoylthio)benzamides [49], acyl perthiols [50,51], arylthioamides [52], 1,2,4- thiadiazolidin-3,5-diones [53], iminothioethers [54], mercaptopyruvate [55], dithioates [56], isothiocyanate [57,58], and thiocarbamates [59] were developed and evaluated for their H2S-releasing properties. Each of these donors release H2S with different mechanisms, triggered by hydrolysis, pH modulation, cellular thiols, photo activation, enzymatic reaction, or others.

2.1. Sulﬁde Salts

Over the years, sulfide salts, such as sodium sulfide (Na2S) and hydrosulfide (NaHS), have been widely used in biological studies to explore the therapeutic prospects of exogenous source of H2S. In particular, these salts showed protective effects against many disease states, such as inflammation [60,61], acute lung injury [62,63], oxidative stress in human neuroblastoma cells [64], Alzheimer’s disease (AD) [65], atherosclerosis [66], and ulcer [67,68]. In addition to these effects, aqueous NaSH solutions delivered in aortic rings led to a 60% greater relaxation over controls, confirming the vasorelaxant properties of H2S[28]. Moreover, several groups demonstrated that the use of sulfide salts was able to induce either pre- and post-conditioning cardioprotection [25,69–73]. Antioxidants 2021, 10, x FOR PEER REVIEW 4 of 27

In addition to these effects, aqueous NaSH solutions delivered in aortic rings led to a 60% greater relaxation over controls, confirming the vasorelaxant properties of H2S [28]. Moreover, several groups demonstrated that the use of sulfide salts was able to induce either pre- and post-conditioning cardioprotection [25,69–73]. Exogenous NaHS has proven to be useful in attenuating ischemia-reperfusion injury [74], protecting against myocardial infarction (MI) [75] and hemorrhagic shock [76]. Furthermore, Calvert et al. reported that long-term Na2S administration attenuates ischemia-induced heart failure, by reducing oxidative stress [24]. The results suggested that the H2S therapy attenuates left ventricular (LV) dilation and cardiac hypertrophy, and leads to an improvement in cardiac function. Solutions of these sulfide salts are prepared by using hydrates of sodium hydrogen Antioxidants 2021, 10, 429 4 of 27 sulfide (NaHS × nH2O), nonahydrate disodium salt (Na2S × 9 H2O), or the anhydrous form. ExogenousIn water NaHS or hasaqueous proven to solution, be useful in attenuatingthese sulfide ischemia-reperfusion salts quickly in- hydrolyze, establishing a dynamicjury [74], protecting equilibrium against myocardial among infarction sulfide (MI) ions [75] and (S hemorrhagic2−), bisulfide shock ions [76]. (HS−), and molecular hydro- Furthermore, Calvert et al. reported that long-term Na2S administration attenuates genischemia-induced sulfide (H heart2S) failure,[77] (Figure by reducing 2); oxidative the ratio stress [24of]. thes The resultse species suggested depends on different parame- that the H2S therapy attenuates left ventricular (LV) dilation and cardiac hypertrophy, and ters,leads tosuch an improvement as temperature, in cardiac function. pressure, and pH. SolutionsAs the of thesepH sulfideincreases, salts are preparedthe H by2S using dissociates hydrates of sodiuminto hydrogenits ions. sul- At a pH below 6, H2S is the fide (NaHS × nH O), nonahydrate disodium salt (Na S × 9 H O), or the anhydrous form. 2 2 2 − predominantIn water or aqueous sulfur solution, species; these at sulfide the physiological salts quickly hydrolyze, pH, establishing H2S and HS species are both present ina dynamicsolution equilibrium in the amongratio sulfideof 1:3; ions at (S a2 −pH), bisulfide of ~9 ions HS (HS− is− ),fully and molecular formed, while at a pH above 15, S2− hydrogen sulfide (H2S) [77] (Figure2); the ratio of these species depends on different isparameters, the predominant such as temperature, species. pressure, and pH.

Figure 2. Mechanism of H2S release from Na2S(1a) and NaHS (1b) in aqueous solution and its Figuredynamic equilibrium2. Mechanism among different of H2 speciesS release (2). from Na2S (1a) and NaHS (1b) in aqueous solution and its dynamic equilibrium among different species (2). As the pH increases, the H2S dissociates into its ions. At a pH below 6, H2S is the − predominant sulfur species; at the physiological pH, H2S and HS species are both present − 2.2.in solution Naturally in the ratio Occurring of 1:3; at a pHDonors of ~9 HS is fully formed, while at a pH above 15, S2− is the predominant species. 2 2.2. NaturallyCurrently Occurring available Donors H S-releasing compounds can be divided into two groups: natu-

rallyCurrently occurring available donors H2S-releasing and compoundssynthetic can donors. be divided Among into two groups: the natural natu- source, allium family and cruciferousrally occurring donors vegetables and synthetic are donors. recognized Among the naturalto be source,rich in allium organosulfur family and compounds. cruciferous vegetables are recognized to be rich in organosulfur compounds. InIn particular, particular, garlic contains garlic at leastcontains thirty-three at least sulfur compounds,thirty-three a concentration sulfur compounds, a concentration higherhigher than than any otherany allium other species. allium The organosulfidesspecies. The in the or alliumganosulfides family are repre- in the allium family are repre- sented by oil-soluble polysulfides and water-soluble thiosulfinates and appear to be respon- sentedsible for many by garlic’soil-soluble medicinal polysulfides effects. It has been demonstratedand water-soluble that the allicin, onethiosulfinates of the and appear to be re- sponsiblebiologically active for componentsmany garlic’s in garlic, andmedicinal its principal transformationeffects. It has products, been which demonstrated that the allicin, are organic polysulfides, provide a critical role in garlic-induced cardioprotection [78–81]. one Inof fact, the it hasbiologically been demonstrated active that components these compounds arein responsiblegarlic, and for lowering its principal transformation prod- the arterial blood pressure, reducing the serum cholesterol and triglycerides, inhibiting the ucts,platelet which aggregation, are preventing organic the polysulfides, atherosclerosis, increasing provide the fibrinolytic a critical activity, role and in garlic-induced cardiopro- tectionreducing the[78–81]. oxidative stress [82]. Among Cruciferae, vegetables such as broccoli, watercress, mustard, and garden cress areIn richfact, in isothiocyanates,it has been suchdemonstrated as sulforaphane (SFN,that highlythese present compounds in broccoli), are responsible for lowering theallyl arterial isothiocyanate blood (AITC, pressure, highly present reducing in black mustard), the benzylseru isothiocyanatem cholesterol (BITC, and triglycerides, inhibiting highly present in garden cress), 4-hydroxybenzyl isothiocyanate (HBITC, highly present in thewhite platelet mustard), andaggregation, erucin (ERU, mainly preventing present in broccoli the athe and rocket)rosclerosis, (Figure3). increasing the fibrinolytic activity, andRecently, reducing it has been the demonstrated oxidative that these stress secondary [82]. metabolites of Brassicaceae, derived from corresponding glucosinolates upon the enzymatic action of myrosinase, showAmong H2S-releasing Cruciferae, properties. This vegetables finding shed light such on the as mechanism broccoli, of actionwatercress, of mustard, and garden cressBrassicaceae are rich and on in the isothiocyanates, role of hydrogen sulfide assuch relevant as playersulforaphane for their nutraceutical (SFN, highly present in broccoli), and pharmaceutical effects [47]. allyl isothiocyanate (AITC, highly present in black mustard), benzyl isothiocyanate (BITC, highly present in garden cress), 4-hydroxybenzyl isothiocyanate (HBITC, highly present in white mustard), and erucin (ERU, mainly present in broccoli and rocket) (Figure 3). Antioxidants 2021, 10, x FOR PEER REVIEW 5 of 27

Antioxidants 2021, 10, 429 5 of 27 Antioxidants 2021, 10, x FOR PEER REVIEW 5 of 27

Figure 3. Chemical structures of natural isothiocyanates, which are abundant in cruciferous vegetables.

Recently, it has been demonstrated that these secondary metabolites of Brassicaceae, derived from corresponding glucosinolates upon the enzymatic action of myrosinase, show H2S-releasing properties. This finding shed light on the mechanism of action of Bras- sicaceae and on the role of hydrogen sulfide as relevant player for their nutraceutical and Figure 3. Chemical structures of natural isothiocyanates, which are abundant in cruciferous vege- Figurepharmaceutical 3. Chemical effects structures [47]. of natural isothiocyanates, which are abundant in cruciferous vegetables. tables. BasedBased onon thethe mechanismmechanism ofof release,release, thethe aboveabove compoundscompounds belongbelong toto thiol-triggeredthiol-triggered donors;donors;Recently, inin fact,fact, it they theyhas beenreactreact demonstrated withwith thethe thiolthiol groupsgroupsthat these ofof glutathioneglutathionesecondary metabolites (GSH)(GSH) oror cysteinecysteine of Brassicaceae, toto releaserelease free H2S. derivedfree H2S. from corresponding glucosinolates upon the enzymatic action of myrosinase, In the intact garlic, the primary sulfur compounds are the γ-glutamyl-S-alk(en)yl-L- showIn H2 theS-releasing intact garlic, properties. the primary This finding sulfur shed compounds light on the are mechanism the γ-glutamyl-S-alk(en)yl- of action of Bras- sicaceaeL-cysteinescysteines and which whichon the can canrole be be hydrolyzedof hydrolyzedhydrogen and sulfide and oxidized oxidized as relevant to toyield yield player alliin. alliin. for Alliinase their Alliinase nutraceutical accounts accounts for and forthe pharmaceuticaltheconversion conversion of alliin effects of alliin to [47].allicin. to allicin. The latter The is latter highly is highlyinstable instable in aqueous in aqueous media and media instantly and instantlydecomposes,Based decomposes, on formingthe mechanism formingsome oil-soluble of some release, oil-soluble compound the above compounds,s, compounds such as suchdiallyl belong as sulfide diallyl to thiol-triggered sulfide(DAS), (DAS),diallyl donors;diallyldisulfide disulfidein (DADS),fact, they (DADS), diallylreact with diallyltrisulfide the trisulfide thiol (DATS), groups (DATS), and of glutathione ajoene and ajoene [43]. (GSH) These [43]. Theseor allicin-derived cysteine allicin-derived to release compounds led to H2S generation upon reaction with endogenous thiols, such as L-cysteine freecompounds H2S. led to H2S generation upon reaction with endogenous thiols, such as L-cysteine andand GSH.InGSH. the intact garlic, the primary sulfur compounds are the γ-glutamyl-S-alk(en)yl-L- cysteinesFurthermore,Furthermore, which can γγ be-glutamyl-S-alk(en)yl-L-cysteines-glutamyl-S-alk(en)yl-L-cysteines hydrolyzed and oxidized to yield can can alliin. be be also Alliinase also converted converted accounts to water-sol- to for water- the conversionsolubleuble organosulfur organosulfur of alliin compoundsto allicin. compounds The including latter including is highlyS-allyl S-allyl instablecysteine cysteine in (SAC) aqueous (SAC) and media andS-allyl S-allyl andmercaptocys- instantly mercap- decomposes,tocysteineteine (SAMC) (SAMC) forming (Figure (Figure some4). 4 ).oil-soluble compounds, such as diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), and ajoene [43]. These allicin-derived compounds led to H2S generation upon reaction with endogenous thiols, such as L-cysteine and GSH. Furthermore, γ-glutamyl-S-alk(en)yl-L-cysteines can be also converted to water-soluble organosulfur compounds including S-allyl cysteine (SAC) and S-allyl mercaptocys- teine (SAMC) (Figure 4).

FigureFigure 4.4. ChemicalChemical structuresstructures ofof commonlycommonly studiedstudied organosulfurorganosulfur compoundscompounds ofof garlic.garlic.

SAC,SAC, aa sulfur-containingsulfur-containing aminoamino acid,acid, waswas consideredconsidered anan endogenousendogenous HH22S-producingS-producing agent by providing the substrate for CSE in the H S synthesis (Figure S1). agent by providing the substrate for CSE in the H22S synthesis (Figure S1). It is responsible for many important activities, such as antioxidative [83,84], anticancer [85,86], anti-hepatotoxic [87,88], and cardioprotective activities [89]. In particular, it showed a cardiovascular protective role in MI by decreasing lipid peroxide products Figureand improving 4. Chemical the structures antioxidant of commonly status. Indeed,studied organosulfur Chuah et al. compounds also demonstrated of garlic. that SAC could upregulate the expression of CSE, leading to enhanced H2S release. The increased SAC, a sulfur-containing amino acid, was considered an endogenous H2S-producing agent by providing the substrate for CSE in the H2S synthesis (Figure S1). Antioxidants 2021, 10, x FOR PEER REVIEW 6 of 27

It is responsible for many important activities, such as antioxidative [83,84], anticancer [85,86], anti-hepatotoxic [87,88], and cardioprotective activities [89]. In particular, Antioxidants 2021, 10, 429 6 of 27 it showed a cardiovascular protective role in MI by decreasing lipid peroxide products and improving the antioxidant status. Indeed, Chuah et al. also demonstrated that SAC could upregulate the expression of CSE, leading to enhanced H2S release. The increased HH2S2S concentrationconcentration in in the the myocardial myocardial and and plasma plasma is responsibleis responsible for for cardioprotection cardioprotection during dur- acuteing acute MI [90 MI]. [90]. Moreover,Moreover, it it was was reported reported that that S-propargyl-cysteine S-propargyl-cysteine (SPRC) (SPRC) and and S-propyl-cysteine S-propyl-cysteine (SPC)(SPC) (Figure (Figure S1) S1) also also exhibited exhibited cardioprotective cardioprotective effects, effects, via via CSE/H CSE/H22SS pathwaypathway [[91,92].91,92]. AmongAmong thethe oil-solubleoil-soluble componentscomponents ofof garlic, garlic, the the major major organosulfur organosulfur compounds compounds werewere reported reported to to be be DADS DADS and and DATS. DATS. In In 2007, 2007, Benavides Benavides et et al. al. demonstrated demonstrated that that these these compoundscompounds could could release release H 2HS2 rapidlyS rapidly through through a thiol-dependent a thiol-dependent mechanism mechanism [93 ]([93]Figure (Figure5 ). In5). the In presencethe presence of GSH, of GSH, DADS DADS can reactcan react with with it to generateit to generate S-allyl S-allyl glutathione glutathione (GSA) (GSA) and theand intermediate the intermediate allyl perthiolallyl perthiol (ASSH), (ASSH), which which reacts reacts with anotherwith another GSH toGSH produce to produce H2S andH2S S-allyland S-allyl glutathione glutathione disulﬁde disulfide (GSSA). (GSSA). Furthermore, Furthermore, the latter the latter compound compound could could also undergoalso undergoα-carbon α-carbon nucleophilic nucleophilic substitution, substitution, generating generating H2S rapidly H2S rapidly [78] (Figure [78] (Figure5a). This 5a). mechanismThis mechanism has been has widelybeen wi conﬁrmeddely confirmed [33,94 –[33,94–99],99], except except for the for rate the of rate H2S of release H2S release from DADS.from DADS. Regarding Regarding this topic, this topic, in fact, in fact, Liang Liang and and co-workers co-workers demonstrated demonstrated that that DADS DADS α releasesreleases H H2S2S very very slowly slowly through through α-carbon-carbon nucleophilic nucleophilic substitution. substitution. The The explanation explanation of of thethe previous previous misunderstanding misunderstanding of of DADS DADS as as a a fast fast H H2S2S donor donor is is DATS DATS contamination contamination in in commercialcommercial samples samples of of DADS; DADS; in in fact, fact, among among these these compounds, compounds, DATS DATS is is responsible responsible for for the rapid H S release. the rapid H2 2S release.

Figure 5. H2S production from organic polysulfides by thiol reactions. Proposed mechanism of Figure 5. H2S production from organic polysulfides by thiol reactions. Proposed mechanism of H2S productionH2S production from (froma) diallyl (a) diallyl disulfide disulfide (DADS) (DADS) and (b and) diallyl (b) diallyl trisulfide trisulfide (DATS). (DATS).

RegardingRegarding DATS DATS (Figure (Figure5b), 5b), two two possible possible thiol thiol−disulfide−disulfide exchange exchange reaction reaction pathways path- canways occur. can Firstlyoccur. (pathwayFirstly (pathway 1), the nucleophilic 1), the nucleophilic attack of GSHattack on of allylic GSH sulfuron allylic of DATS sulfur can of generateDATS can GSSA generate and ASSH,GSSA and with ASSH, the latter with able the tolatter be reduced able to be by reduced GSH and by thus GSH releasing and thus Hreleasing2S; on the H other2S; on sidethe other (pathway side (pathway 2), the nucleophilic 2), the nucleophilic attack of attack GSH onof GSH the central on the sulfurcentral atomsulfur of atom DATS of canDATS lead can to lead the to production the produc oftion ASH of ASH and GSSSA.and GSSSA. The The latter latter can can generate gener- Hate2S H rapidly.2S rapidly. Currently,Currently, isothiocyanates isothiocyanates are are also also drawing drawing a lota lot of of attention attention for for their their many many biological biologi- andcal pharmacologicaland pharmacological effects effects in the in prevention the prevention of important of important human human diseases, diseases, such as cancer,such as neurodegenerativecancer, neurodegenerative processes, processes, and cardiovascular and cardiovascular diseases [diseases100]. In particular,[100]. In particular, Martelli etMartelli al. demonstrated et al. demonstrated that some that selected some arylselect isothiocyanatesed aryl isothiocyanates were able were to release able to H release2S in aH biological2S in a biological environment environment and produce and aproduce vasorelaxant a vasorelaxant effect in rateffect aortic in rings,rat aortic strongly rings, antagonizedstrongly antagonized by a specific by Kv7-blocker, a specific Kv7-blocker, and in the coronary and in the vascular coronary bed, vascular causing bed, an increase causing ofan basal increase coronary of basal flow coronary [57]. Furthermore, flow [57]. Furthermore, the H2S-releasing the H2 propertiesS-releasing of properties the secondary of the metabolites of Brassicaceae, isothiocyanates, are also derived from the rapid reaction with thiols, such as cysteine (Figure6). Antioxidants 2021, 10, x FOR PEER REVIEW 7 of 27

Antioxidants 2021, 10, 429 7 of 27 secondary metabolites of Brassicaceae, isothiocyanates, are also derived from the rapid reaction with thiols, such as cysteine (Figure 6).

Figure 6. The mechanism of H2S release from isothiocyanates. Figure 6. The mechanism of H2S release from isothiocyanates.

The formed Cys-ITCThe formed adduct Cys-ITC undergo adduct intramolecular undergo intramolecular cyclization followed cyclization by followed releas- by releasing organic amine R−NH2 and raphanusamic acid (RA), on one hand, and H2S and 2-car- ing organic amine R−NH2 and raphanusamic acid (RA), on one hand, and H2S and 2-carbylamino-4,5-dihydrothiazole-4-carboxylicbylamino-4,5-dihydrothiazole-4-carboxylic acid, on theacid, other on the hand other [101 hand]. [101].

2.3. Synthetic Donors2.3. Synthetic Donors Although naturally occurring H2S donors represent an attractive tool for in vivo stud- Although naturally occurring H2S donors represent an attractive tool for in vivo studies, their poories, toxicitytheir poor and toxicity the tendency and the oftendency these compounds of these compounds to also generate to also generate some some byproducts, which are not related to H2S production, led researchers to develop novel syn- byproducts, which are not related to H2S production, led researchers to develop novel thetic H2S donors with more favorable pharmacokinetic and safety profiles. synthetic H2S donors with more favorable pharmacokinetic and safety proﬁles. Based on their mechanism of H2S release, we have classified the synthetic compounds Based on their mechanism of H2S release, we have classiﬁed the synthetic compounds Antioxidants 2021, 10, x FOR PEER REVIEW into different groups: hydrolysis-triggered donors, pH-controllable8 of 27 H2S donors, thiol-trig- Antioxidants 2021, 10, x FOR PEER REVIEW into different groups: hydrolysis-triggered donors, pH-controllable H82 Sof donors,27 thiol- Antioxidants 2021, 10, x FOR PEER REVIEW gered donors, enzyme-triggered donors, and others (Table8 1). of 27 Antioxidants 2021, 10, x FOR PEER REVIEW triggered donors, enzyme-triggered donors, and others (Table1). 8 of 27 Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 27 2.3.1. Hydrolysis-Triggered Donors Table 1. H2S-releasingTable 1.moleculesH2S-releasing in cardiovascular molecules in system. cardiovascular system. Table 1. H2S-releasing molecules inMorpholin-4-ium-4-methoxyphenyl(morpho cardiovascular system. lino)phosphinodithioate (GYY4137), a Table 1. H2S-releasing molecules in cardiovascular system. Table 1. H2S-releasing moleculeswater-soluble in cardiovascular Lawesson’s system.Mechanism reagent derivative of (Figure S2), is one of the first slow-releasing Table 1. H2S-releasing molecules in cardiovascular system.Mechanism of Drug Development H2S DonorsH2S Donors Chemical Chemical Structure Mechanism of DrugDrug Development Development Phases 2 H S Release H2S Donors Chemical StructureH S donors developed [40,41,102]MechanismH2 2S release and of the Drugmost commonlyDevelopmentPhases used research tool to investi- H2S Donors Chemical Structure MechanismH2S release of Drug DevelopmentPhases H2S Donors Chemical Structuregate the role of H2S in theMechanismH biological2S release systems.of Drug DevelopmentPhases H2S Donors Chemical Structure H2S release Phases GYY4137 was synthesizedHydrolysis-H2S release starting fromPharmacological LawePhasessson’s reagent, previously obtained by Lawesson’sLawesson’s reagent reagent Hydrolysis-triggeredHydrolysis- Pharmacological Pharmacological studies Lawesson’s reagent heating a mixture of anisoleHydrolysis-triggered with phosphorus Pharmacological pentasulfidestudies (P4S10) [103,104], which reacts Lawesson’s reagent Hydrolysis-triggered Pharmacologicalstudies Lawesson’s reagent with morpholine in dichloromethaneHydrolysis-triggered at roomPharmacological temperaturestudies (Figure S3) [40]. Lawesson’s reagent triggered studies Much evidence has revealedtriggered that GYY4137 exhibitsstudies anti-inflammatory, antioxidant, and anticancer propertiesHydrolysis- [52]. GYY4137 Hydrolysis- Preclinical trials GYY4137GYY4137 It also activatesHydrolysis-triggered vascularHydrolysis-triggered smooth musclePreclinical KATP channels, Preclinical trials and trials relaxes rat aortic rings GYY4137 Hydrolysis-triggered Preclinical trials GYY4137 and renal blood vessels, showingHydrolysis-triggered its anti-hypertensivePreclinical activity trials [40]. GYY4137 triggered Preclinical trials Moreover, GYY4137 hastriggered been reported to inhibit microvascular thrombus formation Hydrolysis- Pharmacological Phosphorodithioates and to stabilize atherosclerosisHydrolysis- plaque by interferingPharmacological with platelet activation and adhesion Phosphorodithioates Hydrolysis-triggered Pharmacologicalstudies PhosphorodithioatesPhosphorodithioates molecule-mediated Hydrolysis-triggeredaggregationHydrolysis-triggered [105]. It alsoPharmacological protectsstudies Pharmacological against diabetic studies myocardial I/R in- Phosphorodithioates Hydrolysis-triggered Pharmacologicalstudies Phosphorodithioates jury, through activation ofHydrolysis- triggeredthe PHLPP-1/Akt/Nrf2 Pharmacologicalstudies pathway [106]. 1,2-dithiole-3-thiones (DTTs) Hydrolysis-triggered Pharmacologicalstudies 1,2-dithiole-3-thiones (DTTs) It has been demonstratedHydrolysis-triggered that GYY4137 Pharmacological slowlystudies releases H 2S upon hydrolysis [40]. In 1,2-dithiole-3-thiones (DTTs) Hydrolysis-triggered Pharmacologicalstudies 1,2-dithiole-3-thiones1,2-dithiole-3-thiones (DTTs) (DTTs) 2015, Alexander et al.Hydrolysis-triggered carefullyHydrolysis-triggered studied the hydrPharmacologicalolysisstudies Pharmacological kinetics of G studiesYY4137, monitoring it 1,2-dithiole-3-thiones (DTTs) triggered studies by a combination of NMRtriggered spectroscopy and massstudies spectrometry [107]. Firstly, a sulfur– JK donors oxygen exchange with waterpH-controlled occurs, leading toPreclinical the release trials of H 2S. The formed product, an JK donors pH-controlled Preclinical trials JK donors aryl-phosphonamidothioate,pH-controlled undergoes completePreclinical hydrolysis trials to release a second molecule JK donors pH-controlled Preclinical trials JK donorsJK donors of H 2S (Figure S4). pH-controlledpH-controlled Preclinical Preclinical trials trials

Phase I and II clinical Ammonium tetrathiomolybdate Phase I and II clinical Ammonium tetrathiomolybdate (NH4)2MoS 4 pH-controlled Phase I andtrials II clinical Ammonium(ATTM) tetrathiomolybdate (NH4)2MoS4 pH-controlled Phase I andtrials II clinical Ammonium(ATTM) tetrathiomolybdate (NH4)2MoS4 pH-controlled Phasefor breast I andtrials IIcancer clinical Ammonium(ATTM) tetrathiomolybdate (NH4)2MoS4 pH-controlled for breasttrials cancer (ATTM) (NH4)2MoS4 pH-controlled for breasttrials cancer (ATTM) Pharmacologicalfor breast cancer N-Benzoylthiobenzamides Thiol-triggered Pharmacologicalfor breast cancer N-Benzoylthiobenzamides Thiol-triggered Pharmacologicalstudies N-Benzoylthiobenzamides Thiol-triggered Pharmacologicalstudies N-Benzoylthiobenzamides Thiol-triggered Pharmacologicalstudies N-Benzoylthiobenzamides Thiol-triggered studies Acyl perthiols Thiol-triggered Preclinicalstudies trials Acyl perthiols Thiol-triggered Preclinical trials Acyl perthiols Thiol-triggered Preclinical trials Acyl perthiols Thiol-triggered PharmacologicalPreclinical trials DithioperoxyanhydridesAcyl perthiols Thiol-triggered PharmacologicalPreclinical trials Dithioperoxyanhydrides Thiol-triggered Pharmacologicalstudies Dithioperoxyanhydrides Thiol-triggered Pharmacologicalstudies Dithioperoxyanhydrides Thiol-triggered Pharmacologicalstudies DithioperoxyanhydridesArylthioamides Thiol-triggered Preclinicalstudies trials Arylthioamides Thiol-triggered Preclinicalstudies trials Arylthioamides Thiol-triggered Preclinical trials Arylthioamides Thiol-triggered Preclinical trials Arylthioamides Thiol-triggered PharmacologicalPreclinical trials Arylisothiocyanates Thiol-triggered Pharmacological Arylisothiocyanates Thiol-triggered Pharmacologicalstudies Arylisothiocyanates Thiol-triggered Pharmacologicalstudies Arylisothiocyanates Thiol-triggered Pharmacologicalstudies Arylisothiocyanates Thiol-triggered Pharmacologicalstudies 1,2,4-Thiadiazolidine-3,5-diones Thiol-triggered Pharmacologicalstudies 1,2,4-Thiadiazolidine-3,5-diones Thiol-triggered Pharmacologicalstudies 1,2,4-Thiadiazolidine-3,5-diones Thiol-triggered Pharmacologicalstudies 1,2,4-Thiadiazolidine-3,5-diones Thiol-triggered Pharmacologicalstudies 1,2,4-Thiadiazolidine-3,5-diones Thiol-triggered Phase studiesI clinical trial Phase studiesI clinical trial Phase I clinical trial SG-1002 Thiol-triggered Phasecomplete I clinical for heart trial SG-1002 Thiol-triggered Phasecomplete I clinical for heart trial SG-1002 Thiol-triggered completefailure for heart SG-1002 Thiol-triggered completefailure for heart SG-1002 Thiol-triggered completefailure for heart failure Pharmacologicalfailure Trimethyl lock (TML) Enzyme-triggered Pharmacological Trimethyl lock (TML) Enzyme-triggered Pharmacologicalstudies Trimethyl lock (TML) Enzyme-triggered Pharmacologicalstudies Trimethyl lock (TML) Enzyme-triggered Pharmacologicalstudies Trimethyl lock (TML) Enzyme-triggered studies studies N-Thiocarboxyanhydrides N-Thiocarboxyanhydrides Enzyme-triggered Preclinical trials N-Thiocarboxyanhydrides(NTAs) Enzyme-triggered Preclinical trials N-Thiocarboxyanhydrides(NTAs) Enzyme-triggered Preclinical trials N-Thiocarboxyanhydrides(NTAs) R Enzyme-triggered Preclinical trials (NTAs) S R Enzyme-triggered Preclinical trials (NTAs) S R O S N H R Enzyme-triggered O O S N R Enzyme-triggered PeroxyTCM B H Preclinical trials PeroxyTCM O O S N Enzyme-triggeredROS-triggered Preclinical trials OB O HN PeroxyTCM O H Enzyme-triggeredROS-triggered Preclinical trials OB O N Enzyme-triggered PeroxyTCM O H ROS-triggered Preclinical trials PeroxyTCM O OB ROS-triggered Preclinical trials OB ROS-triggeredBicarbonate- Pharmacological Thioamino acids O Bicarbonate- Pharmacological Bicarbonate- Pharmacological Thioamino acids Bicarbonate-triggered Pharmacologicalstudies Thioamino acids Bicarbonate-triggered Pharmacologicalstudies Thioamino acids triggered studies Thioamino acids triggered studies triggered studies Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 27 Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 27 Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 27 Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 27 Antioxidants Antioxidants 20212021,, 1010,, xx FORFOR PEERPEER REVIEWREVIEW 88 ofof 2727 Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 27 Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 27 Antioxidants 2021, 10, x FOR PEER REVIEWTable 1. H2S-releasing molecules in cardiovascular system. 8 of 27 Table 1. H2S-releasing molecules in cardiovascular system. Table 1. H2S-releasing molecules in cardiovascular system. Table 1. H2S-releasing molecules in cardiovascular system. Table 1. H2S-releasing molecules in cardiovascular system.Mechanism of Drug Development H2S Donors Table 1. H 2S-releasing Chemical molecules Structure in cardiovascular system.Mechanism of Drug Development H2S Donors Table 1. H2S-releasing Chemical molecules Structure in cardiovascular system.MechanismH 2S release of Drug DevelopmentPhases H2S Donors Table 1. H2S-releasing Chemical molecules Structure in cardiovascular system.Mechanism 2 of Drug Development 2 MechanismH S release of Drug DevelopmentPhases H2S Donors Table 1. H S-releasing Chemical molecules Structure in cardiovascular system.MechanismMechanismH2 S release ofof DrugDrug DevelopmentDevelopmentPhases H2S Donors Chemical Structure H 2S Donors Chemical Structure MechanismH2S release of Drug DevelopmentPhases H2S Donors Chemical Structure MechanismHHydrolysis-H22SS releaserelease of DrugPharmacological DevelopmentPhasesPhases Lawesson’sH2S Donors reagent Chemical Structure MechanismHydrolysis-H2S release of DrugPharmacological DevelopmentPhases Lawesson’sH2S Donors reagent Chemical Structure HHydrolysis-triggered2S release PharmacologicalPhasesstudies Lawesson’s reagent Hydrolysis-Htriggered2S release PharmacologicalstudiesPhases Lawesson’s reagent Hydrolysis-Hydrolysis-triggered PharmacologicalPharmacologicalstudies Lawesson’sLawesson’s reagentreagent Hydrolysis-triggered Pharmacologicalstudies Lawesson’s reagent Hydrolysis-triggeredtriggered Pharmacologicalstudiesstudies Lawesson’s reagent Hydrolysis-triggered Pharmacologicalstudies Lawesson’sGYY4137 reagent Hydrolysis-triggered Preclinicalstudies trials GYY4137 Hydrolysis-triggered Preclinicalstudies trials GYY4137 Hydrolysis-triggered Preclinical trials GYY4137 Hydrolysis-Hydrolysis-triggered Preclinical trials GYY4137GYY4137 Hydrolysis-triggered PreclinicalPreclinical trialstrials GYY4137 Hydrolysis-triggeredtriggered Preclinical trials GYY4137 Hydrolysis-triggered PharmacologicalPreclinical trials PhosphorodithioatesGYY4137 Hydrolysis-triggered PharmacologicalPreclinical trials Phosphorodithioates Hydrolysis-triggered Pharmacologicalstudies Phosphorodithioates Hydrolysis-triggered Pharmacologicalstudies Phosphorodithioates Hydrolysis-Hydrolysis-triggered PharmacologicalPharmacologicalstudies PhosphorodithioatesPhosphorodithioates Hydrolysis-triggered Pharmacologicalstudies 1,2-dithiole-3-thionesPhosphorodithioatesAntioxidants 2021 (DTTs), 10, 429 Hydrolysis-triggeredtriggered Pharmacologicalstudiesstudies 8 of 27 1,2-dithiole-3-thionesPhosphorodithioates (DTTs) Hydrolysis-triggered Pharmacologicalstudies 1,2-dithiole-3-thionesPhosphorodithioates (DTTs) Hydrolysis-triggeredtriggered Pharmacologicalstudies 1,2-dithiole-3-thiones (DTTs) Hydrolysis-Hydrolysis-triggered PharmacologicalPharmacologicalstudies 1,2-dithiole-3-thiones1,2-dithiole-3-thiones (DTTs)(DTTs) Hydrolysis-triggered Pharmacologicalstudies 1,2-dithiole-3-thiones (DTTs) Hydrolysis-triggered Pharmacologicalstudies 1,2-dithiole-3-thiones (DTTs) Hydrolysis-triggered Pharmacologicalstudies 1,2-dithiole-3-thionesJK donors (DTTs) Table 1. Cont. pH-controlledtriggered Preclinicalstudies trials JK donors pH-controlledtriggered Preclinicalstudies trials JK donors pH-controlledtriggered Preclinicalstudies trials JK donors pH-controlled Preclinical trials JKJK donorsdonors MechanismpH-controlledpH-controlled of PreclinicalPreclinical trialstrials JK donorsH2 S Donors Chemical Structure pH-controlled PreclinicalDrug Development trials Phases JK donors pH-controlledH2S Release PhasePreclinical I and II trials clinical AmmoniumJK tetrathiomolybdate donors pH-controlled PhasePreclinical I and II trials clinical Ammonium tetrathiomolybdateAmmonium (NH4)2MoS 4 pH-controlled Phase PhaseI andtrials III and clinical II clinical trials Ammonium tetrathiomolybdate (NH 4)2MoS 4 pH-controlled Phase I and II clinical (ATTM)tetrathiomolybdate (ATTM) (NH4)2MoS4 pH-controlled Phase I andtrialsfor II breastclinical cancer Ammonium(ATTM) tetrathiomolybdate (NH4)2MoS 4 pH-controlled Phasefor breast I andtrials IIcancer clinical Ammonium(ATTM) tetrathiomolybdate (NH4)2MoS 4 pH-controlled Phasefor breastI andtrials IIcancer clinical Ammonium(ATTM) tetrathiomolybdate (NH(NH44))22MoSMoS44 pH-controlledpH-controlled Phasefor breastI andtrialstrials IIcancer clinical Ammonium(ATTM)(ATTM) tetrathiomolybdate (NH4)2MoS4 pH-controlled PhasePharmacologicalfor breastI andtrials IIcancer clinical AmmoniumN-Benzoylthiobenzamides(ATTM) N-Benzoylthiobenzamidestetrathiomolybdate (NH4)2MoS4 Thiol-triggeredThiol-triggeredpH-controlled Pharmacologicalforfor breastbreast Pharmacologicaltrials cancercancer studies N-Benzoylthiobenzamides(ATTM) (NH4)2MoS4 Thiol-triggeredpH-controlled Pharmacologicalfor breaststudiestrials cancer N-Benzoylthiobenzamides(ATTM) Thiol-triggered forPharmacological breaststudies cancer N-Benzoylthiobenzamides Thiol-triggered PharmacologicalforPharmacological breaststudies cancer N-BenzoylthiobenzamidesN-Benzoylthiobenzamides Thiol-triggeredThiol-triggered Pharmacologicalstudies N-BenzoylthiobenzamidesAcyl perthiols Thiol-triggered PharmacologicalPreclinicalstudiesstudies trials N-BenzoylthiobenzamidesAcyl perthiolsAcyl perthiols Thiol-triggeredThiol-triggered PharmacologicalPreclinicalstudies Preclinical trials trials N-BenzoylthiobenzamidesAcyl perthiols Thiol-triggered Preclinicalstudies trials Acyl perthiols Thiol-triggered PharmacologicalPreclinicalstudies trials DithioperoxyanhydridesAcylAcyl perthiolsperthiols Thiol-triggeredThiol-triggered PharmacologicalPreclinicalPreclinical trialstrials DithioperoxyanhydridesAcyl perthiols Thiol-triggered PharmacologicalPreclinicalstudies trials DithioperoxyanhydridesAcyl Dithioperoxyanhydridesperthiols Thiol-triggeredThiol-triggered PharmacologicalPreclinicalstudies Pharmacological trials studies DithioperoxyanhydridesAcyl perthiols Thiol-triggered PharmacologicalPharmacologicalPreclinicalstudies trials DithioperoxyanhydridesDithioperoxyanhydridesArylthioamides Thiol-triggeredThiol-triggered PharmacologicalPreclinicalstudies trials Dithioperoxyanhydrides Thiol-triggered Pharmacologicalstudies DithioperoxyanhydridesArylthioamides Thiol-triggered PharmacologicalPreclinicalstudies trials DithioperoxyanhydridesArylthioamides Thiol-triggered Preclinicalstudies trials ArylthioamidesArylthioamides Thiol-triggeredThiol-triggered PharmacologicalPreclinicalstudies Preclinical trials trials ArylisothiocyanatesArylthioamidesArylthioamides Thiol-triggeredThiol-triggered PharmacologicalPreclinicalPreclinicalstudies trialstrials ArylisothiocyanatesArylthioamides Thiol-triggered PharmacologicalPreclinicalstudies trials ArylisothiocyanatesArylthioamides Thiol-triggered PharmacologicalPreclinicalstudies trials ArylisothiocyanatesArylthioamides Thiol-triggered PharmacologicalPreclinicalstudies trials ArylisothiocyanatesArylisothiocyanates Thiol-triggeredThiol-triggered Pharmacological Pharmacological studies Arylisothiocyanates Thiol-triggered Pharmacologicalstudies 1,2,4-Thiadiazolidine-3,5-dionesArylisothiocyanates Thiol-triggered Pharmacologicalstudiesstudies 1,2,4-Thiadiazolidine-3,5-dionesArylisothiocyanates Thiol-triggered Pharmacologicalstudies 1,2,4-Thiadiazolidine-3,5-dionesArylisothiocyanates Thiol-triggered Pharmacologicalstudies 1,2,4-Thiadiazolidine-3,5-diones1,2,4-Thiadiazolidine Thiol-triggered Pharmacologicalstudies 1,2,4-Thiadiazolidine-3,5-diones Thiol-triggeredThiol-triggered Pharmacologicalstudies Pharmacological studies 1,2,4-Thiadiazolidine-3,5-diones-3,5-diones Thiol-triggered PhasePharmacological studiesI clinical trial 1,2,4-Thiadiazolidine-3,5-diones Thiol-triggered PhasePharmacological studiesstudiesI clinical trial 1,2,4-Thiadiazolidine-3,5-dionesSG-1002 Thiol-triggered PhasecompletePharmacological studiesI clinical for heart trial 1,2,4-Thiadiazolidine-3,5-dionesSG-1002 Thiol-triggered Phasecomplete studiesI clinical for heart trial SG-1002 Thiol-triggered PhasePhasecomplete studiesIfailureI clinicalclinical for heart trialtrial SG-1002 Thiol-triggered PhasecompletePhase Ifailure clinical Ifor clinical heart trial trial complete SG-1002 SG-1002 Thiol-triggeredThiol-triggered Phasecomplete Ifailure clinical for heart trial SG-1002 Thiol-triggered Phasecomplete I clinical for heartheart trial failure SG-1002 Thiol-triggered completefailure for heart SG-1002 Thiol-triggered completePharmacologicalfailurefailure for heart TrimethylSG-1002 lock (TML) Enzyme-triggeredThiol-triggered completePharmacologicalfailure for heart Trimethyl lock (TML) Enzyme-triggered Pharmacologicalstudiesfailure Trimethyl lock (TML) Enzyme-triggered Pharmacologicalstudiesfailure Trimethyl lock (TML) Enzyme-triggered Pharmacologicalstudies Trimethyl lock (TML) Enzyme-triggered Pharmacological TrimethylTrimethyl lock (TML) lock (TML) Enzyme-triggeredEnzyme-triggered Pharmacologicalstudies Pharmacological studies N-ThiocarboxyanhydridesTrimethyl lock (TML) Enzyme-triggered Pharmacologicalstudiesstudies Trimethyl lock (TML) Enzyme-triggered Pharmacologicalstudies N-ThiocarboxyanhydridesTrimethyl lock (TML) Enzyme-triggered Preclinicalstudies trials N-Thiocarboxyanhydrides(NTAs) Enzyme-triggered Preclinicalstudies trials N-Thiocarboxyanhydrides(NTAs) Enzyme-triggered Preclinical trials N-Thiocarboxyanhydrides(NTAs) R Enzyme-triggered Preclinical trials S Enzyme-triggered Preclinical trials N-Thiocarboxyanhydrides(NTAs)N-Thiocarboxyanhydrides R Enzyme-triggered Preclinical trials (NTAs) S R N-Thiocarboxyanhydrides(NTAs)(NTAs) O N Enzyme-triggeredEnzyme-triggered Preclinical Preclinical trials trials S R Enzyme-triggered N-Thiocarboxyanhydrides(NTAs) (NTAs) O NH R Enzyme-triggered Preclinical trials PeroxyTCM O S R Enzyme-triggered Preclinical trials (NTAs) B O S NH R Enzyme-triggered Preclinical trials PeroxyTCM O SS Enzyme-triggered Preclinical trials (NTAs) B H R ROS-triggered O O O N Enzyme-triggered PeroxyTCM B O S NH R Enzyme-triggeredROS-triggered Preclinical trials O O OO S NHN Enzyme-triggered PeroxyTCM O B H R Enzyme-triggeredROS-triggered Preclinical trials PeroxyTCM O OB O S NH ROS-triggered Preclinical trials PeroxyTCM O B H Enzyme-triggeredROS-triggered Preclinical trials OB O N Enzyme-triggeredROS-triggered PeroxyTCM O O H Enzyme-triggeredROS-triggeredBicarbonate- PharmacologicalPreclinical trials O OB O N Enzyme-triggered ThioaminoPeroxyTCM acidsPeroxyTCM B H ROS-triggeredBicarbonate- PharmacologicalPreclinicalPreclinical trials trials ThioaminoPeroxyTCM acids O O ROS-triggeredROS-triggeredBicarbonate-triggered PharmacologicalPreclinicalstudies trials Thioamino acids OB ROS-triggeredBicarbonate-triggered Pharmacologicalstudies Thioamino acids O Bicarbonate-Bicarbonate-triggered PharmacologicalPharmacologicalstudies Thioamino acids Bicarbonate-triggered Pharmacologicalstudies AntioxidantsThioamino 2021, 10, x FOR acids PEER REVIEW Bicarbonate-triggered Pharmacologicalstudies 9 of 27 AntioxidantsThioamino 2021, 10, x FOR acids PEER REVIEW Bicarbonate-triggered Pharmacologicalstudies 9 of 27 Thioamino acids triggered studies ThioaminoThioamino acids acids Bicarbonate-triggeredtriggered studies Pharmacological studies triggered studies

CSE/eNOS- ZYZ803 ZYZ803 CSE/eNOS-dependentCSE/eNOS- Preclinical Preclinical trials trials ZYZ803 dependent Preclinical trials dependent

Zofenopril ACE inhibitor Clinical use for CVD ZofenoprilZofenopril ACEACE inhibitor inhibitor Clinical use Clinical for CVD use for CVD

ACS 14 HS–ASA hybrid Preclinical trials ACS 14 HS–ASA hybrid Preclinical trials

Mithocondrial- AP-39 Mithocondrial- Preclinical trials AP-39 targeted Preclinical trials targeted

Despite GYY4137 having been proven to be a useful biological tool, it suffers from Despite GYY4137 having been proven to be a useful biological tool, it suffers from some drawbacks, such as its contamination with traces of dichloromethane residual from some drawbacks, such as its contamination with traces of dichloromethane residual from crystallization and the slow hydrolysis rate. These aspects could make the attribution of crystallization and the slow hydrolysis rate. These aspects could make the attribution of biological effects to GYY4137-derived H2S uncertain, because of the possible simultaneous biological effects to GYY4137-derived H2S uncertain, because of the possible simultaneous metabolization of dichloromethane to CO, which has biological effects like H2S [108]. metabolization of dichloromethane to CO, which has biological effects like H2S [108]. Therefore, structural modifications of GYY4137 were designed and the resulting an- Therefore, structural modifications of GYY4137 were designed and the resulting analogs were studied. Park and coworkers developed a series of O-aryl- and alkyl-substi- alogs were studied. Park and coworkers developed a series of O-aryl- and alkyl-substituted phosphorodithioates as H2S donors, by replacing the P-C bond in GYY4137 with O- tuted phosphorodithioates as H2S donors, by replacing the P-C bond in GYY4137 with O- substitution [42] (Figure S2). Their studies evidenced that the gaseous release from these substitution [42] (Figure S2). Their studies evidenced that the gaseous release from these novel H2S donors did not significantly improve. In fact, similarly to the parent compound novel H2S donors did not significantly improve. In fact, similarly to the parent compound GYY4137, O-arylated donors showed slow H2S production, whereas O-alkylated donors GYY4137, O-arylated donors showed slow H2S production, whereas O-alkylated donors exhibited very weak H2S generation. exhibited very weak H2S generation. Another class of compounds belonging to the family of hydrolysis-triggered H2S do- Another class of compounds belonging to the family of hydrolysis-triggered H2S donors are 1,2-dithiole-3-thiones (DTTs) (Figure S2). nors are 1,2-dithiole-3-thiones (DTTs) (Figure S2). DTT compounds are anethole dithiolethione (ADT) and its O-demethylated deriva- DTT compounds are anethole dithiolethione (ADT) and its O-demethylated derivative ADT-OH [5-(p-hydroxyphenyl)-3H-1,2-dithiole-3-thione], which were largely used as tive ADT-OH [5-(p-hydroxyphenyl)-3H-1,2-dithiole-3-thione], which were largely used as H2S donors (Figure S5). H2S donors (Figure S5). To obtain DTTs, different methods can be applied. The most used synthetic strategy To obtain DTTs, different methods can be applied. The most used synthetic strategy provides dehydrogenation and sulfurization of an allylic methyl group, by treating it with provides dehydrogenation and sulfurization of an allylic methyl group, by treating it with elemental sulfur or phosphorus pentasulfide products [109] (Figure S6). Alternatively, β- elemental sulfur or phosphorus pentasulfide products [109] (Figure S6). Alternatively, β- ketoesters could react with Lawesson’s reagent to give the desired DTTs [110,111] (Figure ketoesters could react with Lawesson’s reagent to give the desired DTTs [110,111] (Figure S6). S6). To date, several groups have studied the biological effects of ADT and ADT-OH, ob- To date, several groups have studied the biological effects of ADT and ADT-OH, observing an important activity related to the H2S-releasing properties of these compounds. serving an important activity related to the H2S-releasing properties of these compounds. ADT is an FDA-approved drug, which can stimulate bile secretion, restoring salivation ADT is an FDA-approved drug, which can stimulate bile secretion, restoring salivation and relieving dry mouth in patients affected by chemotherapy-induced xerostomia [112]. and relieving dry mouth in patients affected by chemotherapy-induced xerostomia [112]. Additionally, its derivative, ADT-OH, resulted in being useful for reducing cell viability Additionally, its derivative, ADT-OH, resulted in being useful for reducing cell viability via inhibition of histone deacetylase [113,114] and NF-kB activation [115]. via inhibition of histone deacetylase [113,114] and NF-kB activation [115]. Although their H2S-releasing mechanism is still not completely defined, it is widely Although their H2S-releasing mechanism is still not completely defined, it is widely accepted that the production of H2S from DTTs occurs via hydrolysis [34,116,117] (Figure accepted that the production of H2S from DTTs occurs via hydrolysis [34,116,117] (Figure S7). Indeed, it has been demonstrated that upon heating to 120 °C in aqueous solution, S7). Indeed, it has been demonstrated that upon heating to 120 °C in aqueous solution, dithiolethione derivatives gradually release H2S, converting into the corresponding 1,2- dithiolethione derivatives gradually release H2S, converting into the corresponding 1,2- dithiole-3-one [39]. dithiole-3-one [39]. Interestingly, H2S donor hybrids were obtained by coupling the OH group of ADT- Interestingly, H2S donor hybrids were obtained by coupling the OH group of ADT- OH with some commercially available drugs and the resulting compounds were studied OH with some commercially available drugs and the resulting compounds were studied for their H2S-releasing properties and therapeutic effects [118]. for their H2S-releasing properties and therapeutic effects [118]. Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 27

Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 27 Antioxidants 2021, 10, 429 CSE/eNOS- 9 of 27 ZYZ803 Preclinical trials dependent

CSE/eNOS- ZYZ803 Preclinical trials Table 1. Cont. dependent Zofenopril ACE inhibitor Clinical use for CVD Mechanism of H S Donors Chemical Structure Drug Development Phases 2 H S Release Zofenopril ACE inhibitor2 Clinical use for CVD

ACS 14 ACS 14 HS–ASAHS–ASA hybrid hybrid Preclinical Preclinical trials trials ACS 14 HS–ASA hybrid Preclinical trials

Mithocondrial-Mithocondrial- AP-39 AP-39AP-39 Mithocondrial-targetedPreclinicalPreclinical trials Preclinical trials trials targetedtargeted

Despite GYY4137 having been proven to be a useful biological tool, it suffers from Despitesome drawbacks,2.3.1. GYY4137 Hydrolysis-Triggered such having as its contamination been proven Donors with to traces be ofa usefuldichloromethane biological residual tool, from it suffers from some crystallizationdrawbacks,Morpholin-4-ium-4-methoxyphenyl(morpholino)phosphinodithioate andsuch the as slow its hydrolysiscontamination rate. These with aspects traces could of dichloromethanemake the attribution ofresidual from (GYY4137), a biological effects to GYY4137-derived H2S uncertain, because of the possible simultaneous crystallizationwater-soluble and the slow Lawesson’s hydrolysis reagent rate. These derivative aspects (Figure could S2), make is one the of attribution the first slow-releasing of metabolization of dichloromethane to CO, which has biological effects like H2S [108]. biological effects to GYY4137-derived H2S uncertain, because of the possible simultaneous Therefore,H2S donors structural developed modifications [40,41 of, G102YY4137] and were the designed most commonly and the resulting used researchan- tool to investigate metabolization of dichloromethane to CO, which has biological effects like H2S [108]. alogs werethe rolestudied. of HPark2S inand the coworkers biological deve systems.loped a series of O-aryl- and alkyl-substi- Therefore,tuted phosphorodithioates structuralGYY4137 wasmodifications as H synthesized2S donors, by of replacing startingGYY4137 the from wereP-C Lawesson’sbond designed in GYY4137 and reagent, with the O- resulting previously an- obtained by substitution [42] (Figure S2). Their studies evidenced that the gaseous release from these alogs were heatingstudied. a Park mixture and of coworkers anisole with deve phosphorusloped a series pentasulfide of O-aryl- (P4S10) and alkyl-substi- [103,104], which reacts novel H2S donors did not significantly improve. In fact, similarly to the parent compound tuted phosphorodithioates as H2S donors, by replacing the P-C bond in GYY4137 with O- GYY4137,with O-arylated morpholine donors in showed dichloromethane slow H2S production, at room whereas temperature O-alkylated (Figure donors S3) [40]. substitutionexhibited [42] veryMuch (Figure weak evidenceH2 SS2). generation. Their has studies revealed evidenced that GYY4137 that the exhibits gaseous anti-inflammatory, release from these antioxidant, novel H2SAnother donorsand anticancer class did of not compounds significantly properties belonging [52 improve.]. to the family In fact, of hydrolysis-triggered similarly to the Hparent2S do- compound nors are 1,2-dithiole-3-thiones (DTTs) (Figure S2). GYY4137, O-arylatedIt also donors activates showed vascular slow smooth H2S production, muscle K whereaschannels, O-alkylated and relaxes donors rat aortic rings DTT compounds are anethole dithiolethione (ADT) and its O-demethylatedATP deriva- exhibitedtive ADT-OH veryand weak renal [5-(p -hydroxyphenyl)-3H-1,2-dithiole-3-thione],H blood2S generation. vessels, showing its anti-hypertensive which were largely activity used [40 as]. AnotherH2S donors class (FigureMoreover, of compoundsS5). GYY4137 belonging has been to reported the family to inhibitof hydrolysis-triggered microvascular thrombus H2S do- formation nors are 1,2-dithiole-3-thionesToand obtain to DTTs, stabilize different atherosclerosis methods(DTTs) can(Figure be plaque applied. S2). by The interfering most used synthetic with platelet strategy activation and adhesion DTTprovides compoundsmolecule-mediated dehydrogenation are anethole and sulfurization aggregation dithiolethione of an [105 allylic]. (ADT) methyl It also group,and protects its by O-demethylated treating against it with diabetic deriva- myocardial I/R elemental sulfur or phosphorus pentasulfide products [109] (Figure S6). Alternatively, β- tive ADT-OHketoestersinjury, [5-(couldp through-hydroxyphenyl)-3H-1,2-dithiole-3-thione], react with activation Lawesson’s ofreagent the PHLPP-1/Akt/Nrf2to give the desired DTTs which [110,111] pathway were (Figure [ largely106 ]. used as H2S donorsS6). (FigureIt has S5). been demonstrated that GYY4137 slowly releases H2S upon hydrolysis [40]. In To obtainTo2015, date, DTTs, several Alexander different groups ethave al.methods studied carefully the can biological studied be applied. effects the hydrolysis Theof ADT most and usedADT-OH, kinetics synthetic ob- of GYY4137, strategy monitoring providesserving dehydrogenationit an by important a combination activity and related ofsulfurization NMR to the spectroscopyH2S-releasing of an allylic properties and methyl mass of these spectrometrygroup, compounds. by treating [107]. it Firstly,with a sulfur– ADT isoxygen an FDA-approved exchange drug, with which water can occurs, stimulate leading bile secretion, to the restoring release ofsalivation H S. The formedβ product, an elementaland relieving sulfur dryor phosphorusmouth in patients pentasulfide affected by chemotherapy-induced products [109] (Figure xerostomia S6). [112].Alternatively,2 - ketoestersAdditionally, couldaryl-phosphonamidothioate, reactits derivative, with Lawesson’s ADT-OH, resulted reagent undergoes in being to give useful complete the for desired reducing hydrolysis DTTs cell viability to[110,111] release a(Figure second molecule S6). via inhibitionof H2 Sof (Figure histone deacetylase S4). [113,114] and NF-kB activation [115]. To date,Although severalDespite their groups H2S-releasing GYY4137 have mechanism studied having beenthe is still biological proven not completely toeffects be defined, a usefulof ADT it is biological andwidely ADT-OH, tool, itob- suffers from acceptedsome that the drawbacks, production suchof H2S asfrom its DTTs contamination occurs via hydrolysis with traces [34,116,117] of dichloromethane (Figure residual from servingS7). an Indeed, important it has been activity demonstrated related thatto the upon H 2heatingS-releasing to 120 properties°C in aqueous of solution, these compounds. crystallization and the slow hydrolysis rate. These aspects could make the attribution of ADT isdithiolethione an FDA-approved derivatives graduallydrug, which release can H2S, stimulate converting bileinto thesecretion, corresponding restoring 1,2- salivation and relievingdithiole-3-onebiological dry [39]. mouth effects in patients to GYY4137-derived affected by chemotherapy-induced H2S uncertain, because xerostomia of the possible [112]. simultaneous Additionally,Interestingly,metabolization its derivative, H2S donor of ADT-OH, dichloromethanehybrids were resulted obtained to inby CO, beingcoupling which useful the has OH for biologicalgroup reducing of ADT- effects cell viability like H2S[ 108]. OH with some commercially available drugs and the resulting compounds were studied via inhibition ofTherefore, histone deacetylase structural [113,114] modifications and NF-kB of GYY4137 activation were [115]. designed and the resulting for their H2S-releasing properties and therapeutic effects [118]. Althoughanalogs their wereH2S-releasing studied. mechanism Park and coworkersis still not completely developed defined, a series it of is O-aryl-widely and alkyl- substituted phosphorodithioates as H S donors, by replacing the P-C bond in GYY4137 accepted that the production of H2S from DTTs occurs2 via hydrolysis [34,116,117] (Figure S7). Indeed,with it has O-substitution been demonstrated [42] (Figure that S2). upon Their heating studies to evidenced120 °C in aqueous that the gaseoussolution, release from these novel H S donors did not significantly improve. In fact, similarly to the parent com- dithiolethione derivatives2 gradually release H2S, converting into the corresponding 1,2- dithiole-3-onepound [39]. GYY4137, O-arylated donors showed slow H2S production, whereas O-alkylated donors exhibited very weak H2S generation. Interestingly, H2S donor hybrids were obtained by coupling the OH group of ADT- OH with some commerciallyAnother class available of compounds drugs and belonging the resulting to the compounds family of hydrolysis-triggered were studied H2S donors are 1,2-dithiole-3-thiones (DTTs) (Figure S2). for their H2S-releasing properties and therapeutic effects [118]. DTT compounds are anethole dithiolethione (ADT) and its O-demethylated derivative ADT-OH [5-(p-hydroxyphenyl)-3H-1,2-dithiole-3-thione], which were largely used as H2S donors (Figure S5). Antioxidants 2021, 10, 429 10 of 27

To obtain DTTs, different methods can be applied. The most used synthetic strategy provides dehydrogenation and sulfurization of an allylic methyl group, by treating it with elemental sulfur or phosphorus pentasulfide products [109] (Figure S6). Alterna- tively, β-ketoesters could react with Lawesson’s reagent to give the desired DTTs [110,111] (Figure S6). To date, several groups have studied the biological effects of ADT and ADT-OH, observing an important activity related to the H2S-releasing properties of these compounds. ADT is an FDA-approved drug, which can stimulate bile secretion, restoring salivation and relieving dry mouth in patients affected by chemotherapy-induced xerostomia [112]. Additionally, its derivative, ADT-OH, resulted in being useful for reducing cell viability via inhibition of histone deacetylase [113,114] and NF-kB activation [115]. Although their H2S-releasing mechanism is still not completely defined, it is widely accepted that the production of H2S from DTTs occurs via hydrolysis [34,116,117](Figure S7). Indeed, it has been demonstrated that upon heating to 120 ◦C in aqueous solution, dithiolethione derivatives gradually release H2S, converting into the corresponding 1,2-dithiole- 3-one [39]. Antioxidants 2021, 10, x FOR PEER REVIEWInterestingly, H S donor hybrids were obtained by coupling the OH group of ADT-OH10 of 27 2 with some commercially available drugs and the resulting compounds were studied for their H2S-releasing properties and therapeutic effects [118]. TheyThey areare usually usually synthesized synthesized by by coupling coupling the the active active compounds compounds with with ADT-OH ADT-OH in the in presencethe presence of N,N’-dicyclohexylcarbodiimide of N,N’-dicyclohexylcarbodiimide (DCC) and(DCC) 4-dimethylaminopyridine and 4-dimethylaminopyridine (DMAP) (Figure S8) [119]. These H S donor hybrids exhibited their efficacy in many pharmaco- (DMAP) (Figure S8) [119]. 2These H2S donor hybrids exhibited their efficacy in many phar- logical fields, such as inflammation [120,121] (H2S-donating diclofenac, ACS-15/ATB- macological fields, such as inflammation [120,121] (H2S-donating diclofenac, ACS- 337; H2S-donating mesalamine, ATB-429), cancer [122] (H2S-donating sulindac, HS-SUL; 15/ATB-337; H2S-donating mesalamine, ATB-429), cancer [122] (H2S-donating sulindac, H2S-donating aspirin, HS-ASA/ACS-14; H2S-donating ibuprofen, HS-IBU; H2S-donating HS-SUL; H2S-donating aspirin, HS-ASA/ACS-14; H2S-donating ibuprofen, HS-IBU; H2S- naproxen, HS-NAP), erectile dysfunction [123] (H2S-donating sildenafil, ACS 6), neu- donating naproxen, HS-NAP), erectile dysfunction [123] (H2S-donating sildenafil, ACS 6), rodegeneration (H2S-donating latanoprost, ACS 67, for glaucoma treatment [124], and neurodegeneration (H2S-donating latanoprost, ACS 67, for glaucoma treatment [124], and H2S-donating levodopa, ACS 83, for Parkinson’s disease [125]), and cardioprotection H2S-donating levodopa, ACS 83, for Parkinson’s disease [125]), and cardioprotection (H2S- (H S-donating aspirin, HS-ASA/ACS 14, and AP-39) (Figure7). donating2 aspirin, HS-ASA/ACS 14, and AP-39) (Figure 7).

FigureFigure 7.7. ChemicalChemical structuresstructures ofof HH22SS donordonor hybridshybrids involvedinvolved inin cardiovascularcardiovascular disease.disease.

Indeed,Indeed, RossoniRossoni and and co-workers co-workers demonstrated demonstrated that that ACS14 ACS14 exerts exerts strong strong protective protective ef- fectseffects against against buthionine buthionine sulfoximine sulfoximine (BSO)-induced (BSO)-induced cardiovascular cardiovascular diseases, diseases, by increasingby increas- systolicing systolic blood blood pressure pressure and loweringand lowering heart heart rates rates in rats in [ 126rats]. [126]. Additionally, Additionally, oral adminis- oral ad- trationministration of ACS of 14 ACS reduces 14 reduce BSO-induceds BSO-induced hypertensive hypertensive effects, effects, unlike aspirinunlike whichaspirin has which no effect.has no Moreover, effect. Moreover, ACS 14 ACS treatment 14 treatment in BSO in rats BSO reduces rats reduces myocardial myocardial I/R injury I/R injury [126–128 [126–]. 128].More recently, another hybrid H2S donor, named AP-39, resulting from the reaction of triphenylphosphonium with ADT-OH, was developed [129] and studied for its H2S- More recently, another hybrid H2S donor, named AP-39, resulting from the reaction releasing properties and potential pharmacological effects. of triphenylphosphonium with ADT-OH, was developed [129] and studied for its H2S- AP-39 signiﬁcantly inhibits oxidative stress-induced toxicity and protects against releasing properties and potential pharmacological effects. acute cardiac arrest, renal, and myocardial I/R injury, by inhibiting the mitochondrial AP-39 significantly inhibits oxidative stress-induced toxicity and protects against permeability transition pore [130–133]. acute cardiac arrest, renal, and myocardial I/R injury, by inhibiting the mitochondrial permeability transition pore [130–133].

2.3.2. pH-Controllable H2S Donors Starting from GYY4137 structure, Kang and coworkers developed a series of phosphonamidothioates, named JK donors [134]. To synthesize these compounds, the phenylphosphonothioic dichloride was treated with 3-hydroxypropionitrile and, consequently, with a selected C-protected amino acid, such as glycine, phenylalanine, valine, alanine, and proline. These steps provided the intermediate, which underwent LiOH-mediated hydrolysis, furnishing the final compounds (Figure S9). These donors represent the first class of pH-controllable H2S donors. Indeed, in aqueous media under acidic conditions (pH = 2.0–6.0), JK donors had higher H2S release rates, whereas at neutral and mildly basic pH (7.4–8.0), they caused slower and less H2S release. Kang and co-workers observed that these donors undergo a new hydrolysis mechanism of release. The protonation of phosphonamidothioates leads to the formation of the corresponding phosphorothiols, which cyclize via nucleophilic addition of the carboxylic acid group, resulting in breakage of the P-S bond and in release of H2S (Figure 8). Antioxidants 2021, 10, 429 11 of 27

2.3.2. pH-Controllable H2S Donors Starting from GYY4137 structure, Kang and coworkers developed a series of phosphonamidothioates, named JK donors [134]. To synthesize these compounds, the phenylphosphonothioic dichloride was treated with 3-hydroxypropionitrile and, consequently, with a selected C-protected amino acid, such as glycine, phenylalanine, valine, alanine, and proline. These steps provided the intermediate, which underwent LiOH-mediated hydrolysis, furnishing the ﬁnal compounds (Figure S9). These donors represent the ﬁrst class of pH-controllable H2S donors. Indeed, in aqueous media under acidic conditions (pH = 2.0–6.0), JK donors had higher H2S release rates, whereas at neutral and mildly basic pH (7.4–8.0), they caused slower and less H2S release. Kang and co-workers observed that these donors undergo a new hydrolysis mechanism of release. The protonation of phosphonamidothioates leads to the formation of the Antioxidants 2021, 10, x FOR PEER REVIEWcorresponding phosphorothiols, which cyclize via nucleophilic addition of the carboxylic11 of 27

acid group, resulting in breakage of the P-S bond and in release of H2S (Figure8).

Figure 8. H2S-releasing mechanism from JK donors. Figure 8. H2S-releasing mechanism from JK donors.

Moreover,Moreover, the the two two main main donors donors (JK-1 (JK-1 and and JK-2) JK-2) of of the the series series (Figure (Figure S10) S10) showed showed protectiveprotective effects effects on on cellular cellular and and murine murine models model ofs of myocardial myocardial I/R I/R injury. injury. They They were were also also successfulsuccessful in in reducing reducing infarct infarct size size and and circulating circulating troponin-I troponin-I level level signiﬁcantly significantly [134 [134].]. Furthermore,Furthermore, ammonium ammonium tetrathiomolybdate tetrathiomolybdate (ATTM), (ATTM), an an excellent excellent copper copper chelator chelator withwith the the formula formula (NH (NH4)42)MoS2MoS4,4, was was identiﬁed identified as as a a pH-dependent pH-dependent H H2S2S donor donor [ 135[135].]. Indeed, Indeed, itit has has beenbeen demonstrateddemonstrated that that H H2S2S can can be be generated generated from from ATTM ATTM under under strong strong acidic acidic con- conditionsditions (5% (5% H2 HSO2SO4) (Figure4) (Figure S11). S11). Later,Later, Xu Xu and and co-workers co-workers described described ATTM ATTM as a water-solubleas a water-soluble and a and pH-sensitive a pH-sensitive slow- releasingslow-releasing H2S donor H2S donor [136]. More[136]. recently,More recently, it has beenit has found been thatfound ATTM that ATTM exerts protectiveexerts pro- effectstective on effects the heart on the and heart brain and in brain rat models in rat models of myocardial of myocardial and cerebral and cerebral I/R injury I/R injury and attenuatesand attenuates I/R injury I/R injury [137]. [137]. ConsideringConsidering thatthat somesome preclinicalpreclinical data suggested that that H H2S2S is is responsible responsible for for protec- pro- tectivetive effects effects in indoxorubicin- doxorubicin- and and adriamycin adriamycin-induced-induced cardiomyopathy cardiomyopathy during during cancer cancer ther- therapiesapies [138–140] [138–140 and] and that that ATTM ATTM is is currently currently in in clinical clinical trials for thethe treatmenttreatment ofof breast breast cancercancer due due to to its its copper copper depletion depletion effects effects [ 141[141],], the the researchers researchers are are studying studying the the potential potential dualdual role role of of ATTM ATTM as as an an anticancer anticancer and and cardioprotective cardioprotective agent agent in in patients patients with with cancer cancer treatedtreated with with doxorubicin doxorubicin or or adriamycin. adriamycin.

2.3.3.2.3.3. Thiol-Triggered Thiol-Triggered Donors Donors Thiol-activated H S-releasing compounds were some of the ﬁrst synthetic donors to Thiol-activated H2 2S-releasing compounds were some of the first synthetic donors to be reported. They can release H S by reacting with endogenous thiol-containing molecules, be reported. They can release2 H2S by reacting with endogenous thiol-containing mole- such as GSH, which are relatively abundant in mammals. Some of these H S donors were cules, such as GSH, which are relatively abundant in mammals. Some of these2 H2S donors foundwere found to exhibit to exhibit protective protective effects effects in cardiovascular in cardiovascular diseases diseases (Figure (Figure S12). S12). Among the ﬁrst nucleophile-triggered H2S donors, a series of N-(benzoylthio) benza- Among the first nucleophile-triggered H2S donors, a series of N-(benzoylthio)ben- mides were synthesized by Zhao et al. [49] with the goal of developing compounds which zamides were synthesized by Zhao et al. [49] with the goal of developing compounds are stable in aqueous solution. The N-(acylthiol)benzamide derivatives were prepared which are stable in aqueous solution. The N-(acylthiol)benzamide derivatives were pre- by treating thiocarboxylic acids with hydroxylamine-O-sulfonic acid under basic condi- pared by treating thiocarboxylic acids with hydroxylamine-O-sulfonic acid under basic conditions; the following reaction between the obtained S-acylthiohydroxylamines and benzoic anhydride led to obtain the final products (Figure S13). The resulting compounds were proven to be stable in aqueous buffers and to be able to release H2S only following a nucleophilic attack by thiols. The structure–activity relationship studies showed that electron withdrawing groups cause a faster H2S release, unlike electron donating groups. In addition, H2S release from N-(benzoylthio)benzamides was also detected in the plasma, revealing that N-SH compounds have efficacy in complex systems. These compounds protect human keratinocytes against methylglyoxal (MGO)-induced cell damage and dysfunction, which is prevalent among patients with diabetes mellitus [142]. Moreover, they were evaluated in animal models of myocardial I/R injury. The reduction in infarct size over controls in murine models indicated N-(benzoylthio)benzamides as cardioprotective agents [143]. The thiol-triggered mechanism of release from these H2S donors starts with a thioester exchange between the donor and a first molecule of cysteine generating S-benzoyl cysteine and N-mercaptobenzamide, an N-SH intermediate (Figure 9). The resulting S-benzoyl cysteine, due to its high reactivity, undergoes a fast S to N acyl transfer, leading to the formation of a more stable N-benzoyl cysteine. At the same Antioxidants 2021, 10, 429 12 of 27

tions; the following reaction between the obtained S-acylthiohydroxylamines and benzoic anhydride led to obtain the ﬁnal products (Figure S13). The resulting compounds were proven to be stable in aqueous buffers and to be able to release H2S only following a nucleophilic attack by thiols. The structure–activity relationship studies showed that electron withdrawing groups cause a faster H2S release, unlike electron donating groups. In addition, H2S release from N-(benzoylthio)benzamides was also detected in the plasma, revealing that N-SH compounds have efﬁcacy in complex systems. These compounds protect human keratinocytes against methylglyoxal (MGO)-induced cell damage and dysfunction, which is prevalent among patients with diabetes melli-

Antioxidants 2021tus, 10 [, 142x FOR]. Moreover,PEER REVIEW they were evaluated in animal models of myocardial I/R injury. The re- 12 of 27 duction in infarct size over controls in murine models indicated N-(benzoylthio)benzamides as cardioprotective agents [143]. The thiol-triggered mechanism of release from these H2S donors starts with a thioester exchange betweentime, the the donor formed and N-SH a ﬁrst intermediate molecule of reacts cysteine with generating another molecule S-benzoyl of cysteinecysteine to give benzamide and cysteine perthiol, which finally reacts with cysteine to generate cystine and and N-mercaptobenzamide, an N-SH intermediate (Figure9). release H2S.

Figure 9. H2S release from N-benzoylthiobenzamides. Figure 9. H2S release from N-benzoylthiobenzamides.

The resulting S-benzoylAs cysteine cysteine, perthiol due was to itsfound high to reactivity, be the key undergoes intermediate a fast for S toH N2S acylgeneration from transfer, leadingN-mercapto-based to the formation of donors, a more stablein addition N-benzoyl to its already-known cysteine. At the involvement same time, the in H2S biosynthesis catalyzed by CSE [118,144], a library of perthiol-based donors (RC(O)–S–SR’) were formed N-SH intermediate reacts with another molecule of cysteine to give benzamide and designed and developed by Zhao and co-workers [50], aiming to mimic H2S bioproduc- cysteine perthiol, which finally reacts with cysteine to generate cystine and release H S. tion. 2 As cysteine perthiol was found to be the key intermediate for H S generation from N- The authors prepared the above compounds as cysteine2 and penicillamine deriva- mercapto-based donors, in addition to its already-known involvement in H S biosynthesis tives (Figure S14) and protected the unstable –SH residue with2 acyl groups. catalyzed by CSE [118,144], a library of perthiol-based donors (RC(O)–S–SR’) were designed Cys-S-SH-based donors were obtained through the treatment of N-benzoyl cysteine and developed by Zhao and co-workers [50], aiming to mimic H S bioproduction. methyl ester with 2,2′-dipyridyl disulfide to attain a2 reactive intermediate, which then re- The authorsacted prepared with a the thioacid above to compounds produce the as desired cysteine compounds and penicillamine (Figure S15). derivatives (Figure S14) and protectedTo obtain the the unstable other –SHderivatives, residue C- with and acyl N-protected groups. penicillamine was treated with Cys-S-SH-based donors were obtained through the treatment of N-benzoyl cysteine 2,2′-dibenzothioazolyldisulfide,0 furnishing an intermediate, which quickly reacted with methyl ester withdifferent 2,2 -dipyridyl thioacids disulfideto produce to the attain desired a reactive donors intermediate,(Figure S16). which then reacted with a thioacidSurprisingly, to produce it thewas desired found that compounds these donors (Figure exhibit S15). different H2S-releasing profiles. To obtain theIndeed, other in derivatives, the presence C- of and thiols, N-protected penicillamin penicillaminee-based donors was showed treated higher with efficiency in 0 2,2 -dibenzothioazolyldisulfide,H2S production than furnishing cysteine-based an intermediate, donors, which quicklywere able reacted to release with very small different thioacidsamounts to produce of H2S. the Probably, desired the donors two different (Figure S16).behaviors could be explained by the presence Surprisingly,of itthe was two found adjacent that methyl these donors groups exhibit of penicillamine-based different H2S-releasing donors, profiles. which prevent the Indeed, in the presencecleavage of of thiols, the disulfide penicillamine-based bonds by thiols. donors showed higher efficiency in H2S production thanThese cysteine-based donors showed donors, tunable which wererelease able rates to releaseby varying very the small aromatic amounts R substituent. of H2S. Probably,H the2S release two different depends behaviors on both steric could and be explainedelectronic factors, by the presencewith electron-withdrawing of the two sub- adjacent methylstituents groups ofaccelerating penicillamine-based release rates, donors, and bulky which substituents prevent the on cleavagethe aromatic of the ring retarding disulfide bondsrelease by thiols. rates. Regarding the H2S-releasing mechanism, the acyl perthiol donors also required disulfide exchange to allow H2S production, similarly to N-mercapto-based donors; therefore, deacylation and subsequent H2S release occurred (Figure 10). Antioxidants 2021, 10, 429 13 of 27

These donors showed tunable release rates by varying the aromatic R substituent. H2S release depends on both steric and electronic factors, with electron-withdrawing substituents accelerating release rates, and bulky substituents on the aromatic ring retarding release rates. Regarding the H2S-releasing mechanism, the acyl perthiol donors also required disul- Antioxidants 2021ﬁde, 10, x exchange FOR PEER REVIEW to allow H2S production, similarly to N-mercapto-based donors; therefore, 13 of 27

deacylation and subsequent H2S release occurred (Figure 10).

Figure 10. Proposed mechanism of H2S release from perthiol-based donors; R’-SH = cysteine or GSH. Figure 10. Proposed mechanism of H2S release from perthiol-based donors; R’-SH = cysteine or GSH.

H2S release from penicillamine-derived perthiol donors was also studied in vivo and H S release from penicillamine-derived perthiol donors was also studied in vivo and 2 in cardiac myocytes (H9c2 cells), confirming their ability to release. Moreover, the selected in cardiac myocytes (H9c2 cells), confirming their ability to release. Moreover, the selected donors were evaluated into a murine model of myocardial I/R injury. The results proved donors were evaluated into a murine model of myocardial I/R injury. The results proved that they exhibit cardioprotective activity due to their ability to reduce the infarct size. that they exhibit cardioprotective activity due to their ability to reduce the infarct size. Analogous perthiol-based donors are the dithioperoxyanhydrides (RC(O)–S– Analogous perthiol-based donors are the dithioperoxyanhydrides (RC(O)–S–SC(O)R’) [51], SC(O)R’) [51], which also have the disulfide linkage in their structures (Figure S12). which also have the disulfide linkage in their structures (Figure S12). Both alkyl and aromatic dithioperoxyanhydride donors were obtained by two possi- Both alkyl andble synthetic aromatic strategies: dithioperoxyanhydride iodine oxidation donors of the werethiocarboxylat obtainedes by [145] two possibleor reactions between synthetic strategies:thiocarboxylic iodine oxidation acids and of methoxycarbonylsulfenyl the thiocarboxylates [145 chloride] or reactions [146] (Figure between S17). thiocarboxylic acids and methoxycarbonylsulfenyl chloride [146] (Figure S17). H2S release from these compounds was measured amperometrically and found in H S release from these compounds was measured amperometrically and found in both 2 both buffers and cellular lysates, confirming H2S-releasing properties of dithioperoxy- buffers and cellularanhydrides. lysates, confirming When treated H2S-releasing with thiols, properties these donors of dithioperoxyanhydrides. formed a key intermediate for H2S When treated withproduction, thiols, thesethe acyl-persulfides, donors formed which, a key reacting intermediate with the for excess H2S production, of thiols, could directly the acyl-persulfides,release which, H2S and reacting RSSAc with (pathway the excess a, Figure of thiols, 11); couldalternatively, directly it release could Hproduce2S a new and RSSAc (pathwayperthiol a, species Figure 11(RS-SH),); alternatively, which reacting it could in produceturn with a thiols, new perthiolleads to speciesdisulfide formation (RS-SH), whichand reacting H2S release in turn (pathway with thiols, b, Figure leads 11). to disulfide formation and H2S release (pathway b, Figure 11Furthermore,). these donors were found to induce concentration-dependent vaso- Furthermore,relaxation these donors of pre-contracted were found rat to ao inducertic rings, concentration-dependent presumably owing to H2S vasore- release. Moreover, laxation of pre-contractedthe arylthioamides rat aortic (Figure rings, S12) presumably were also classified owing to as H thiol-activated2S release. Moreover, H2S donors [52]. The the arylthioamidesnon-heterocyclic (Figure S12) aromatic were also compounds classified were as thiol-activated obtained by mixing H2S donors the corresponding [52]. ben- The non-heterocycliczonitrile aromatic with P4S compounds10; whereas the were reaction obtained between by mixing amidesthe with corresponding Lawesson’s reagent led to benzonitrile withheterocyclic P4S10; whereas aromatic the reactioncompounds between being amides obtained with (Figure Lawesson’s S18). reagent led to heterocyclic aromaticThese compounds compounds being showed obtained the ability (Figure to release S18). H2S in the presence of thiols, simi- These compoundslarly to showedDADS and the GYY4137, ability to two release slow H-releasing2S in the presencedonors. To of date, thiols, the similarly H2S release mecha- to DADS and GYY4137,nism and tworelative slow-releasing intermediates donors. and final To pr date,oducts the have H2S releasenot yet been mechanism clearly described. and relative intermediates and final products have not yet been clearly described. Antioxidants 2021, 10, 429 14 of 27 Antioxidants 2021, 10, x FOR PEER REVIEW 14 of 27

Figure 11. Proposed mechanism for H2S release from dithioperoxyanhydrides; R′-SH = cysteine or Figure 11. Proposed mechanism for H S release from dithioperoxyanhydrides; R0-SH = cysteine GSH. 2 or GSH.

After confirming their H2S-releasing profile, Calderone and coworkers tested the ef- After confirming their H S-releasing profile, Calderone and coworkers tested the effects of these 2donors on noradrenaline-induced vasoconstriction in isolated rat aortic fects of these donors on noradrenaline-induced vasoconstriction in isolated rat aortic rings. rings. The results suggested that the lead compound, p-hydroxybenzothioamide (Figure p The results suggestedS19), has that protective the lead compound,activity in cardiovascul-hydroxybenzothioamidear systems, inhibiting (Figure norepinephrine-induced S19), has protective activityvasoconstriction in cardiovascular in ex systems, vivo in rat inhibiting aortic rings, norepinephrine-induced and reducing systolic blood vasocon- pressure. striction in ex vivo in rat aortic rings, and reducing systolic blood pressure. Considering the slow and sustained H2S release profile of p-hydroxybenzothioamide Consideringand the its slow ease and of conjugation sustained H to2S other release compou profilends, of p this-hydroxybenzothioamide donor was used to develop many and its ease of conjugationhybrid compounds. to other Among compounds, these, p-hydroxybenzothioamide this donor was used to developwas conjugated many with the ac- hybrid compounds.tive compound Among these, naproxen,p-hydroxybenzothioamide obtaining ATB-346 (Figure was S19) conjugated [147]. Thus, with the the efficacy of this active compoundhybrid naproxen, as an anticancer obtaining drug ATB-346 was investigated, (Figure S19) confirming [147]. Thus, its theapoptotic efficacy action of in human this hybrid as anmelanoma anticancer cells drug [148]. was Moreover, investigated, it has been confirming demonstrated its apoptotic that ATB-346 action reduces in gastro- human melanomaintestinal cells [ 148tract]. injury Moreover, and exhibits it has been chemopreve demonstratedntive action that against ATB-346 colorectal reduces cancer when gastrointestinal tractcompared injury to and naproxen exhibits [149]. chemopreventive Recently, a phase action II study against was colorectal completed cancer and revealed that when comparedATB-346 to naproxen is useful [149 ].for Recently, the treatment a phase of pa II studyin associated was completed with osteoarthritis and revealed [150]. that ATB-346 is usefulThese for thepositive treatment preclinical of pain data associated for ATB-346 with osteoarthritis suggested that [150 H].2S-releasing hybrid These positivedrugs preclinical could undergo data for further ATB-346 development suggested for that the H 2treatmentS-releasing of hybridcardiovascular drugs disease. could undergo furtherMoreover, development Martelli for and the co-workers treatment ofreported cardiovascular aryl isothiocyanates disease. as another series of Moreover, Martellithiol-activated and co-workers H2S donors reported(Figure S12) aryl [57]. isothiocyanates These compounds as another showed series vasorelaxant ef- of thiol-activatedfects H2 onS donors conductance (Figure and S12) coronary [57]. These arteries. compounds showed vasorelaxant effects on conductanceNoteworthy, and coronary 4-carboxyphenyl-isothiocyanate arteries. (4-CPI) and 3-pyridyl-isothiocyanate Noteworthy,(Figure 4-carboxyphenyl-isothiocyanate S20) exhibited cardioprotective (4-CPI) effects and against 3-pyridyl-isothiocyanate I/R injury, reducing myocardial (Figure S20) exhibitedcontractility, cardioprotective ventricular arrhythmias, effects against and I/Roxidative injury, stress reducing [58,151]. myocardial contractility, ventricularThe arrhythmias,mechanism of and H2S oxidative release from stress these [58, 151compounds]. has already been described above (Figure 6). The nature of the R groups supporting the isothiocyanate function have The mechanism of H2S release from these compounds has already been described above (Figure6).a strong The nature impact of on the H2S-releasing R groups supportingrates. For examples, the isothiocyanate p-nitrophenyl function isothiocyanate produces H2S relatively fast, while benzylisothiocyanate and phenethyl isothiocyanate are have a strong impact on H2S-releasing rates. For examples, p-nitrophenyl isothiocyanate considered slow H2S donors [101]. produces H2S relatively fast, while benzylisothiocyanate and phenethyl isothiocyanate are More recently, 1,2,4-thiadiazolidine-3,5-diones (THIA) (Figure S12), which are isothi- considered slow H2S donors [101]. More recently,ocyanate 1,2,4-thiadiazolidine-3,5-diones derivatives, developed by Severino (THIA) (Figureet al. [53], S12), were which demonstrated are isoth- to be novel iocyanate derivatives,thiol-triggered developed H2S bydonors. Severino et al. [53], were demonstrated to be novel These compounds were obtained through oxidative condensation of isothiocyanates thiol-triggered H2S donors. with isocyanates in the presence of SO2Cl2, using both the conventional synthetic strategy, These compounds were obtained through oxidative condensation of isothiocyanates previously described in literature [152], and the microwave-assisted synthesis [53]. The with isocyanates in the presence of SO Cl , using both the conventional synthetic strategy, second way led better yields2 2 being obtained and the reduction of reaction times (Figure previously described in literature [152], and the microwave-assisted synthesis [53]. The S21). The authors clearly defined the mechanism of H2S release by using a combination of second way led better yields being obtained and the reduction of reaction times (Figure S21). HPLC, MS, and NMR to isolate and characterize the intermediates and the final products. The authors clearly defined the mechanism of H2S release by using a combination of HPLC, MS, and NMR to isolate and characterize the intermediates and the final products. Antioxidants 2021, 10, 429 15 of 27

Antioxidants 2021, 10, x FOR PEER REVIEW 15 of 27

As depicted in Figure 12, the nucleophilic attack of thiols, for example cysteine, on the electronAs depicted deﬁcient in Figure sulfur 12, atom the ofnucleophilic the 1,2,4-thiadiazolidine-3,5-dione attack of thiols, for example nucleus cysteine, opens on the ring and produces an intermediate. When this latter reacts with a second molecule the electron deficient sulfur atom of the 1,2,4-thiadiazolidine-3,5-dione nucleus opens the of cysteine, it generates cystine and phenyl(phenylcarbamoyl)carbamothioic S-acid, an ring and produces an intermediate. When this latter reacts with a second molecule of cys- instable intermediate, which is immediately hydrolyzed, yielding 1,3-diphenylurea, CO , teine, it generates cystine and phenyl(phenylcarbamoyl)carbamothioic S-acid, an instable2 and H2S. intermediate, which is immediately hydrolyzed, yielding 1,3-diphenylurea, CO2, and H2S.

Figure 12. H2S-releasing mechanism from 1,2,4-thiadiazolidine-3,5-diones (THIA). Figure 12. H2S-releasing mechanism from 1,2,4-thiadiazolidine-3,5-diones (THIA).

ItIt was was also also found found that that these these donors donors exerted exerted vasodilating vasodilating activity activity in in a a concentrationconcentration- dependentdependent manner manner on on isolated isolated aorta aorta rings. rings. Furthermore,Furthermore, a sodiuma sodium polysulthionate, polysulthionate, named named SG-1002 SG-1002 (Figure (Figure S12), S12), was developed.was devel- Thisoped. synthetic This synthetic H2S prodrug H2S prodrug is a water-insoluble is a water-insoluble microcrystalline microcrystalline material, mostly material, consisting mostly ofconsistingα-sulfur (aboutof α-sulfur 99% S(about8) hydrophilized 99% S8) hydrophilized with traces ofwith ionic traces substances, of ionic suchsubstances, as sodium such sulfate,as sodium sodium sulfate, thiosulfate, sodium thiosulfate, and sodium and polythionates, sodium polythionates, which enhance which its enhance bioavailabil- its bio- ityavailability [153]. [153]. SG-1002SG-1002 was was obtained obtained viavia comproportionationcomproportionation ofof sulfursulfur atomsatoms inin thethe -2-2 andand +4+4 ox-oxi- idationdation states in a strongly acidic acidic medium medium of of high high ionic ionic strength. strength. This This synthetic synthetic route, route, to- togethergether with with properties properties and and some therapeutic applications ofof SG1002SG1002 areare describeddescribed in in a a patentpatent [ 154[154].]. H H22S-releasingS-releasing fromfrom SS88 isis activatedactivated byby thiolsthiolsand and this this reaction reaction with with GSH GSH is is depicteddepicted in in Figure Figure S22 S22 [ 155[155–157].–157]. SG-1002,SG-1002, when when administered administered orally, orally, produced produc aed more a more sustained sustained and consistent and consistent increase in- increase H2S andin H sulfane2S and sulfane sulfur levels sulfur in levels models in models of pressure of pressure overload overload heart failure. heart failure. Moreover, More- it improvedover, it improved coronary coronary artery vascular artery vascular reactivity reac andtivity attenuated and attenuated high-fat diet-inducedhigh-fat diet-induced cardiac hypertrophycardiac hypertrophy and dysfunction and dysfunction [153,158, 159[153,158,159].]. Importantly,Importantly, SG-1002 SG-1002 has has completed completed the the phase phase I clinicalI clinical trial trial where where its its H H2S-donating2S-donating abilityability in in patients patients with with congestive congestive heart heart failure failure was was investigated investigated [ 160[160].]. The The results results of of this this trialtrial confirmed confirmed that that SG-1002 SG-1002 is is useful useful both both in in healthy healthy patients patients and and in in patients patients with with heart heart failure.failure. Indeed, Indeed, it it effectively effectively enhances enhances sulfide sulfide and and NO NO levels levels in in patients patients with with heart heart failure. failure. Nevertheless,Nevertheless, further further trials trials are are required required to to test test the the drug’s drug’s efficacy efficacy [161 [161].]. ToTo date, date, it it is is the the only only sulfide-based sulfide-based therapy therap clinicallyy clinically tested tested in patients in patients with with cardiovas- cardio- cularvascular diseases. diseases. 2.3.4. Enzyme-Triggered Donors 2.3.4. Enzyme-Triggered Donors In recent years, enzyme-triggered H2S donors have been received increasing interest In recent years, enzyme-triggered H2S donors have been received increasing interest due to their many advantages over the other triggers already mentioned above. Among due to their many advantages over the other triggers already mentioned above. Among these, a series of esterase-sensitive prodrugs of H2S, based on a lactonization reaction, also these, a series of esterase-sensitive prodrugs of H2S, based on a lactonization reaction, also known as “trimethyl lock” (TML) [162], was developed by Zheng et al. [163] (Figure S23). known as ‘‘trimethyl lock” (TML) [162], was developed by Zheng et al. [163] (Figure S23). The first synthesized derivative, HP-101, was obtained starting from compound 1 (Figure S24), which, reacting with Lawesson’s reagent under microwave conditions [164], AntioxidantsAntioxidants2021 2021, 10, 10, 429, x FOR PEER REVIEW 16 ofof 2727

Antioxidants 2021, 10, x FOR PEER REVIEW 16 of 27

resultedThe ﬁrstin an synthesized intermediate derivative, that was then HP-101, treated was with obtained one equivalent starting fromof sodium compound hydrox- 1 ide. (Figureresulted S24), in an which, intermediate reacting that with was Lawesson’s then trea reagentted with under one equivalent microwave of conditions sodium hydrox- [164], resultedThe in obtained an intermediate TML-based that wasprodrugs then treatedwere able with to one release equivalent H2S, through of sodium the hydroxide.cleavage of ide. a phenolicThe obtained ester by TML-based an esterase prodrugs and subsequent were able lactonization, to release H owing2S, through to the thesteric cleavage repulsion of The obtained TML-based prodrugs were able to release H2S, through the cleavage of aof phenolic three methyl ester bygroups an esterase (Figure and 13). subsequent lactonization, owing to the steric repulsion a phenolic ester by an esterase and subsequent lactonization, owing to the steric repulsion of three methyl groups (Figure 13). of three methyl groups (Figure 13).

Figure 13. Mechanism of esterase-triggered H2S release.

Figure 13. Mechanism of esterase-triggered H2S release. FigureInterestingly, 13. Mechanism a ofTML esterase-triggered derivative, HP-102 H2S release. (Figure S24), was found to attenuate myocardial I/R injury in a dose-dependent manner and exhibited a cardioprotective action Interestingly, a TML derivative, HP-102 (Figure S24), was found to attenuate myo- [165].Interestingly, Thus, this compound a TML derivative, is currently HP-102 under (Figure investigation S24), was to found better to define attenuate its chemical, myocar- cardial I/R injury in a dose-dependent manner and exhibited a cardioprotective action dialpharmacological, I/R injury in a and dose-dependent safety profile mannerin the hope and that exhibited it could a cardioprotective be tested in clinical action trials. [165 ]. Thus,[165]. this Thus, compound this compound is currently is currently under investigation under investigation to better to define better its define chemical, its chemical, pharma- Another class of enzyme-triggered H2S donors is carbonyl sulfide (COS)-releasing pharmacological, and safety profile in the hope that it could be tested in clinical trials. cological,compounds. and Indeed, safety profile COS is in a the substrate hope that of itca couldrbonic be anhydrase tested in clinical(CA), which trials. catalyzes its AnotherAnother classclass ofof enzyme-triggered enzyme-triggered HH2SS donorsdonors isis carbonyl carbonyl sulfide sulfide (COS)-releasing (COS)-releasing conversion into H2S [166,167]. 2 compounds.compounds. Indeed,Indeed, COSCOS isis aa substratesubstrate ofof carbonic carbonic anhydrase anhydrase (CA), (CA), which which catalyzes catalyzes its its An example of these dual COS/H2S donors are N-thiocarboxyanhydrides (NTAs) conversion into H2S [166,167]. conversion(Figure S25) into [168,169], H2S[166 which,167]. have the advantage of releasing, in addition to COS, only AnAn exampleexample ofof these these dual dual COS/H COS/H2SS donorsdonors areare N-thiocarboxyanhydridesN-thiocarboxyanhydrides (NTAs)(NTAs) innocuous peptide byproducts [170]. 2 (Figure(Figure S25)S25) [168[168,169],,169], which which have have the the advantage advantage ofof releasing, releasing, inin addition addition toto COS, COS, only only The sarcosine NTA derivative (NTA1) (Figure S25) was synthesized starting from innocuousinnocuous peptide peptide byproducts byproducts [ 170[170].]. sarcosine (N-methyl glycine) through the route depicted in Figure S26 [171]. TheThe sarcosinesarcosine NTANTA derivativederivative (NTA1)(NTA1) (Figure (Figure S25) S25) was was synthesized synthesized starting starting fromfrom The mechanism leading to H2S production from NTA1 consists of ring-opening by a sarcosinesarcosine (N-methyl (N-methyl glycine) glycine) through through the the route route depicted depicted in in Figure Figure S26 S26 [ 171[171].]. biological nucleophile, such as an amine. The resulting dipeptide byproducts were con- TheThe mechanismmechanism leadingleading toto HH22S production from from NTA1 NTA1 consists consists of of ring-opening ring-opening by by a firmed by GC-MS and LC-MS, respectively. After COS release, its conversion into H2S abiological biological nucleophile, nucleophile, such such as asan an amine. amine. The The resulting resulting dipeptide dipeptide byproducts byproducts were were con- occurs through the rapid enzymatic action of CA (Figure 14) [170]. confirmedfirmed by byGC-MS GC-MS and and LC-MS, LC-MS, respectively. respectively. After After COS COS release, itsits conversionconversion intointo H HS2S Furthermore, Powell et al. showed that the treatment of endothelial cells with NTA12 occursoccurs throughthrough the the rapid rapid enzymatic enzymatic action action of of CA CA (Figure (Figure 14 14))[ 170[170].]. increased proliferation, which is an important step in the angiogenetic process [170]. Furthermore, Powell et al. showed that the treatment of endothelial cells with NTA1 increased proliferation, which is an important step in the angiogenetic process [170].

Figure 14. Mechanism of carbonyl sulfide (COS)/H2S release from NTA1. Figure 14. Mechanism of carbonyl sulﬁde (COS)/H2S release from NTA1.

Figure 14. Mechanism of carbonyl sulfide (COS)/H2S release from NTA1. Furthermore,Among the class Powell of COS/H et al. showed2S donors, that some the treatment ROS-triggered of endothelial H2S donors cells (peroxyTCM- with NTA1 increased1, 2, 3) were proliferation, developed which[172], using is an importanta ROS-clea stepvable in aryl the angiogeneticboronate as the process protecting [170]. group Among the class of COS/H2S donors, some ROS-triggered H2S donors (peroxyTCM- [173,174]. They were obtained as depicted in Figure S27. 1, 2, 3) were developed [172], using a ROS-cleavable aryl boronate as the protecting group [173,174]. They were obtained as depicted in Figure S27. Antioxidants 2021, 10, 429 17 of 27

Antioxidants 2021, 10, x FOR PEER REVIEW 17 of 27 Among the class of COS/H2S donors, some ROS-triggered H2S donors (peroxyTCM- Antioxidants 2021, 10, x FOR PEER REVIEW 17 of 27 1, 2, 3) were developed [172], using a ROS-cleavable aryl boronate as the protecting group [173,174]. They were obtained as depicted in Figure S27. TheThe obtained obtained compounds compounds werewere found found to to release release H H2S2S inin thethe presencepresence ofof an an oxidant, oxidant, The obtained compounds were found to release H2S in the presence of an oxidant, suchsuch as as H H2O2O22.. InIn thisthis mechanism,mechanism, thethe boronateboronate esterester waswas convertedconverted toto aa phenol phenol by by ROS, ROS, such as H2O2. In this mechanism, the boronate ester was converted to a phenol by ROS, releasingreleasing COS, COS, which which was was quickly quickly converted converted into into H H2S2S by by CA CA [ 172[172]] (Figure (Figure 15 15).). releasing COS, which was quickly converted into H2S by CA [172] (Figure 15).

Figure 15. ProposedFigure mechanism 15. Proposed for Hmechanism2O2-triggered for H COS/H2O2-triggered2S release. COS/H 2S release. Figure 15. Proposed mechanism for H2O2-triggered COS/H2S release.

Moreover,Moreover, itit wasMoreover, demonstrated demonstrated it was demonstratedthat that the the combin combined thated the role rolecombin of boronate ofed boronate role ester, of boronate ester, as ROS as ester, ROSscav- as ROS scavenger, with the produced H2S, as cytoprotective agent, led the ROS-activated H2S donors scavenger,enger, with with the produced the produced H2S, Has2 S,cytoprotective as cytoprotective agent, agent, led the led ROS-activated the ROS-activated H2S donors H2S donorsto exhibit to exhibit importantto important exhibit activities important activities in many activities in manyhuman in human manypathologies human pathologies driven pathologies driven by high driven by oxidative high by oxidative high stress, oxidative stress, such as cardiovascular diseases [133]. stress,such as such cardiovascular as cardiovascular diseases diseases [133]. [133 ]. However, further in vivo studies are required to better characterize the biological However,However, furtherfurtherin in vivo vivostudies studies are are required required to to better better characterize characterize the the biological biological nature of these donors and to confirm their efficacy. naturenature of of these these donors donors and and to to confirm confirm their their efficacy. efficacy. 2.3.5. Others 2.3.5.2.3.5. Others Others In 2012, Zhou et al. reported that thioamino acids, such as thioglycine and thiovaline, InIn 2012, 2012, Zhou Zhou et et al. al. reported reported that that thioamino thioamino acids, acids, such such as as thioglycine thioglycine and and thiovaline, thiovaline, were bicarbonate-triggered H2S donors [48]. They synthesized the selected compounds were bicarbonate-triggered H S donors [48]. They synthesized the selected compounds were bicarbonate-triggeredstarting from commerciallyH2 2S donors [48]. available They Boc-prot synthesizedected aminothe selected acids, whichcompounds were treated with starting from commercially available Boc-protected amino acids, which were treated with starting from commercially1,1′-carbonyldiimidazole available Boc-prot in dichloromethaneected amino andacids, subsequently which were with treated gaseous with H 2S. The fol- 1,10-carbonyldiimidazole in dichloromethane and subsequently with gaseous H S. The 1,1′-carbonyldiimidazolelowing deprotection in dichloromethane with trifluoroacetic and subsequently acid (TFA) with in dichloromethane gaseous H2S.2 The furnished fol- the de- following deprotection with trifluoroacetic acid (TFA) in dichloromethane furnished the lowing deprotectionsired compoundswith trifluoroacetic (Figure S28). acid (TFA) in dichloromethane furnished the de- desired compounds (Figure S28). sired compounds (FigureThe authors S28). proved that, in the presence of bicarbonate, these compounds react TheThe authorsauthorsthrough provedproved their that,that, amine inin the thegroups presence presence and consequently of of bicarbonate, bicarbonate, undergo these these a compounds compoundscyclization, reactconverting react to the through their amine groups and consequently undergo a cyclization, converting to the through their aminecorresponding groups andamino consequently acid N-carboxyanhydrides undergo a cyclization, with the productionconverting of to H the2S (Figure 16). corresponding amino acid N-carboxyanhydrides with the production of H2S (Figure 16). corresponding Consideringamino acid N-carboxyanhydridesthe proposed mechanism with of the releas productione and the of high H2S concentrations (Figure 16). of bicar- Considering the proposed mechanism of release and the high concentrations of bicarbonate Considering thebonate proposed in blood, mechanism thioamino of acids releas coulde and be theeffective high Hconcentrations2S donors. of bicar- in blood, thioamino acids could be effective H2S donors. bonate in blood, thioamino acids could be effective H2S donors.

Figure 16. Proposed mechanism for H2S release from thioamino acids.

Figure 16. Proposed mechanism 2for H2S release from thioamino acids. Figure 16. Proposed mechanismIndeed, H forS-releasing H2S release capabilities from thioamino of thioglycine acids. and thiovaline were evaluated and proved amperometrically. In addition, both thioglycine and thiovaline were found to Indeed, H2S-releasing capabilities of thioglycine and thiovaline were evaluated and proved amperometrically. In addition, both thioglycine and thiovaline were found to Antioxidants 2021, 10, 429 18 of 27

Antioxidants 2021, 10, x FOR PEER REVIEW 18 of 27

Indeed, H2S-releasing capabilities of thioglycine and thiovaline were evaluated and proved amperometrically. In addition, both thioglycine and thiovaline were found to enhanceenhance cGMPcGMP formationformation inin aa concentration-dependent concentration-dependent mannermanner andand induce induce signiﬁcant significant relaxationrelaxation of of pre-contracted pre-contracted mouse mouse aortic aortic rings. rings. However,However, the the high high reactivity reactivity of of these these amino amino acids acids must must be be considered. considered. In In fact, fact, it it was was foundfound that that under under aerobic aerobic conditions conditions they they could could rapidly rapidly undergo undergo amidation amidation [ 175[175–180]–180] and and oxidationoxidation reactions reactions [ 51[51],], yielding yielding products products which which could could lead lead to to unwanted unwanted side side effects. effects. Recently,Recently, a novela novel H2S-NO-releasing H2S-NO-releasing molecule, molecule, 2-(N-Boc-amino)-3-prop-2-ynylsulfanyl- 2-(N-Boc-amino)-3-prop-2-ynyl- propionicsulfanylpropionic acid, named acid, ZYZ803, named was ZYZ803, developed was de [181veloped] by coupling [181] by SPRC coupling with furoxan,SPRC with upon fu- treatmentroxan, upon with treatment Boc anhydride with Boc (Figure anhydride S29) [ 181(Figure,182]. S29) [181,182]. AsAs already already described described above, above, SPRC SPRC could could enhance enhance H2S concentration H2S concentration in plasma in through plasma thethrough activation the activation of CSE enzyme of CSE enzyme [92] while [92] furoxan while furoxan is a thiol-triggered is a thiol-triggered NO-releasing NO-releasing com- poundcompound [183]. [183]. Therefore,Therefore, ZYZ803ZYZ803 stimulated both the the expr expressionession of of CSE CSE and and the the activity activity of ofendo- en- dothelial NO synthase (e-NOS), releasing H S and NO, respectively (Figure 17). These thelial NO synthase (e-NOS), releasing H2S and2 NO, respectively (Figure 17). These gas- gasotransmittersotransmitters promoted promoted angiogenesis angiogenesis thro throughugh the the SIRT1/VEGF/cGMP SIRT1/VEGF/cGMP pathway pathway and and via viacross-talk cross-talk between between STAT3 STAT3 and and CaMKII CaMKII [184]. [184 ].

Figure 17. H2S/NO-releasing mechanism from ZYZ803. Figure 17. H2S/NO-releasing mechanism from ZYZ803.

AsAs reported reported by by Hu Hu et et al., al., ZYZ803 ZYZ803 significantly significantly promoted promoted endothelial endothelial cell cell angiogenesis angiogene- bothsis both in vitro in vitro and and in vivo in vivo [185 [185].]. Indeed,Indeed, thethe oraloral administration administration ofof ZYZ803ZYZ803 inin a a mouse mouse model model of of hindlimb hindlimb ischemia ischemia stimulatedstimulated angiogenesis angiogenesis more more effectively effectively than th individuallyan individually administered administered SPRC SPRC and furoxan, and fu- confirmingroxan, confirming the additive the additive protective protective actions ofactions H2S and of H NO2S and releasing NO releasing from ZYZ803 from [ZYZ803185]. [185].Additionally, ZYZ803 regulated the vascular tone in isolated rat aortic rings [181], attenuatedAdditionally, cardiac dysfunction,ZYZ803 regulated and improved the vascular myocardial tone in injuryisolated after rat heart aortic failure rings [[181],186], exertingattenuated a powerful cardiac dysfunction, cardioprotective and effect.improved myocardial injury after heart failure [186], exertingLastly, a powerful another Hcardioprotective2S donor with activityeffect. in the cardiovascular system is zofenopril, an angiotensin-convertingLastly, another H2S donor enzyme with (ACE) activity inhibitor. in the cardiovascular Undergoing hydrolysis system is inzofenopril, the liver, an it formsangiotensin-converting zofenoprilat, its active enzyme metabolite (ACE) containinginhibitor. Undergoing a thiol group hydrolysis (Figure S30). in the liver, it formsIt iszofenoprilat, important to its underline active metabolite that zofenopril containing is not a a thiol new group drug; in(Figure fact, it S30). was approved for medicalIt is important use in 2000. to underline Recently, that Bucci zofenopril et al. demonstrated is not a new that drug; this in compound fact, it was can approved release Hfor2S[ medical187], which use in mediates 2000. Recently, the beneficial Bucci et effects al. demonstrated of zofenopril, that such this ascompound anti-inflammatory, can release pro-angiogenicH2S [187], which and mediates anti-apoptotic the beneficial activity effects [187–191 of ].zofenopril, such as anti-inflammatory, pro-angiogenicAdditionally, and zofenopril anti-apoptotic was reportedactivity [187–191]. to improve vascular function in a model of hypertension,Additionally, combining zofenopril its ACE was inhibitor reported activity to improve and H 2vascularS-releasing function properties in a [model187]. of hypertension,Indeed, it wascombining also found its ACE that inhibitor zofenopril activity administration and H2S-releasing increases properties the levels [187]. of H2 S metabolitesIndeed, in it thewas plasma also found of mice, that protectingzofenopril againstadministration myocardial increases I/R injury,the levels and of pigs, H2S improvingmetabolites endocardial in the plasma blood of mice, flow inprotecting myocardial against ischemia myocardial [192]. I/R injury, and pigs, improving endocardial blood flow in myocardial ischemia [192].

3. Conclusions Antioxidants 2021, 10, 429 19 of 27

3. Conclusions During last decades, remarkable progress has been made in the understanding of the biological activity of H2S and its mechanism of actions in the cardiovascular system and in the discovering and developing of novel H2S-releasing compounds. Herein we presented an overview of the currently available H2S donating agents that have been tested in preclinical models of cardiovascular diseases, with a focus on fundamental chemistry and their H2S-releasing properties and mechanisms. As described above, these H2S donors include inorganic sulﬁde salts, natural and synthetic organic compounds, characterized by different release triggering mechanisms. Although many donors have been developed and tested in vitro and in vivo studies, showing their ability to release H2S and the positive effects when treating cardiovascular diseases, to date it is not yet possible to identify an ideal donor, owing to some drawbacks for each of them. Indeed, the major limitations for many donors are that the release does not mimic the endogenous H2S production and leads to the simultaneous generation of reactive byproducts, whose nature and biological activity are often still unknown. To overcome these drawbacks, several new classes of donors with a sustained and controlled H2S release were designed, synthesized, and studied for their biological activity, also deﬁning their mechanism of release; moreover, they were successfully tested in various animal models of cardiovascular disease. Among them, at least two H2S-releasing compounds (ATTM and SG-1002) have completed the phase I clinical trials and registered on clinicaltrials.gov for cardiovascular disease and breast cancer (Table1). Nevertheless, there are still too few available donors with oral bioavailability, which is crucial for clinical translation, and being available for testing in in vivo pharmacokinetic studies. Furthermore, the safety and long-term effects of many H2S-releasing compounds have yet to be more tested and fully characterized. Bearing in mind the above considerations, this work, by reviewing the available H2S donors exhibiting cardioprotective activity and thus providing an insightful knowledge of their chemical nature and mechanisms of release, could enhance the chances to design novel H2S-donating molecules useful to help further reduce the risks of cardiovascular disease in the coming years.

Supplementary Materials: The following are available online at https://www.mdpi.com/2076-3 921/10/3/429/s1, The following are available online. Figure S1. The chemical structures of SAC, SPC, and SPRC. Figure S2. Chemical structures of hydrolysis-triggered H2S donors. Figure S3. Chemical synthesis of GYY4137. Figure S4. The hydrolytic degradation of GYY4137 and the H2S release. Figure S5. Structures of the most used DTTs as H2S donors. Figure S6. Different chemical strategies to synthesize DTTs. Figure S7. Mechanism of H2S release from DTTs. Figure S8. Chemical synthesis of HS-NSAIDs. Figure S9. Synthetic strategy to obtain JK donors. Figure S10. Chemical structures of two main JK donors. Figure S11. The H2S release from ammonium tetrathiomolybdate (ATTM). Figure S12. Chemical structures of thiol-triggered H2S donors with cardioprotective activity. Figure S13. Chemical synthesis of N-(benzoylthio)benzamides. Figure S14. Chemical structures of perthiol-based donors. Figure S15. Synthetic route to obtain cysteine perthiol-based donors. Figure S16. Chemical synthesis of penicillamine perthiol-based donors. Figure S17. The synthetic strategies to obtain dithioperoxyanhydride-based H2S. Figure S18. Chemical synthesis of (a) non-heterocyclic and (b) heterocyclic p-hydroxy-arylthio-amides. Figure S19. The thioamides: General structure, chemical structures of lead compound (p-hydroxybenzothioamide) and its hybrid with naproxen (ATB-346). Figure S20. Chemical structures of two aryl isothiocyanates exhibiting cardioprotective activity. Figure S21. Synthesis of THIA. Conventional synthetic strategy: (i) SO2Cl2/diethyl ether, 24 ◦ h; (ii) H2O, reﬂux, 30 min; microwave-assisted synthesis: (i) SO2Cl2/diethyl ether/MW 500 W, 50 C, ◦ 1h; (ii) H2O, MW 500 W, 120 C, 30 min. Figure S22. Mechanism of H2S release from SG1002. Figure S23. Chemical structures of “TML”-based H2S prodrugs HP-101 and HP-102. Figure S24. Synthesis of “TML”-based H2S prodrug HP-101. Figure S25. Chemical structures of N-thiocarboxyanhydrides (NTAs) and sarcosine NTA derivative (NTA1). Figure S26. Chemical synthesis of sarcosine NTA Antioxidants 2021, 10, 429 20 of 27

derivative (NTA1). Figure S27. Chemical structures and synthesis of ROS-activated H2S donors. Figure S28. Synthesis of thioamino acids. Figure S29. Chemical structure and synthesis of ZYZ803. Figure S30. Chemical structures of (a) zofenopril and (b) its active metabolite, zofenoprilat. Author Contributions: Conceptualization, A.C., G.C. and V.S.; resources, F.F. (Francesco Frecentese) and A.S.; writing—original draft preparation, A.C., B.S., E.M. and E.P.; writing—review and editing, B.S. and F.F. (Ferdinando Fiorino); supervision, B.S. and F.F. (Ferdinando Fiorino). All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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