Design, Synthesis and Evaluation of Novel Molecular Hybrids between Antiglaucoma Drugs and H2S Donors

Glaucoma is a group of eye diseases consisting of optic nerve damage with corresponding loss of field vision and blindness. Hydrogen sulfide (H2S) is a gaseous neurotransmitter implicated in various pathophysiological processes. It is involved in the pathological mechanism of glaucomatous neuropathy and exerts promising effects in the treatment of this disease. In this work, we designed and synthetized new molecular hybrids between antiglaucoma drugs and H2S donors to combine the pharmacological effect of both moieties, providing a heightened therapy. Brinzolamide, betaxolol and brimonidine were linked to different H2S donors. The H2S-releasing properties of the new compounds were evaluated in a phosphate buffer solution by the amperometric approach, and evaluated in human primary corneal epithelial cells (HCEs) by spectrofluorometric measurements. Experimental data showed that compounds 1c, 1d and 3d were the hybrids with the best properties, characterized by a significant and long-lasting production of the gasotransmitter both in the aqueous solution (in the presence of L-cysteine) and in the intracellular environment. Because, to date, the donation of H2S by antiglaucoma H2S donor hybrids using non-immortalized corneal cells has never been reported, these results pave the way to further investigation of the potential efficacy of the newly synthesized compounds.


Introduction
The glaucomas are a group of eye diseases characterized by damage of the optic nerve with corresponding loss of field vision [1]. Glaucoma is the leading cause of irreversible blindness and, to date, 11 million people went blind because of this disease [2]. With advancing age, the likelihood of developing glaucoma is higher; therefore, due to the rapid increase in aging population, by 2040 the number of individuals with glaucoma is projected to grow up to 111.8 million [3].
Although the main cause of this neuropathy is unknown and the pathogenesis is not completely understood, intraocular pressure (IOP) is the major modifiable risk factor and is regulated by the balance of aqueous humor (AH) production and outflow. In a healthy human eye, under steady-state conditions, IOP ranges from 10 to 21 mmHg [4]. Usually, in patients with glaucoma, there is an increase in IOP due to a reduced outflow facility of aqueous humor [5].
Glaucomas can be classified into open-angle glaucoma (OAG) and angle-closure glaucoma (ACG) [1,[5][6][7][8][9]. In eyes with open-angle glaucoma, there are no clinically visible perturbations in the eye and the aqueous humor is free to leave the globe; in contrast, with angle-closure glaucoma, the AH drainage is anatomically reduced or blocked [6].
In addition to elevated intraocular pressure, there are common risk factors for the development of glaucomas, such as age, ethnicity, family history of glaucoma, systemic hypertension and diabetes mellitus [5,10,11].
Treatment of glaucoma neuropathies aims to reduce IOP, and on the basis of causes, risk factors, severity and type of glaucoma, different medical options such as topical therapy, oral therapy, surgery or laser procedure are available. First-line treatment consists of the topical application through eye drops of IOP-lowering drugs in monotherapy or as drug combinations. Different classes of medications are used to treat glaucoma, and they either increase the outflow of AH from the eye (prostaglandin analogs and cholinomimetics) or reduce its formation (α 2 -adrenergic agonists, β-adrenergic antagonists and carbonic anhydrase inhibitors) [5,7]. H 2 S is a colorless, flammable and pungent gas and it has been recognized as a third endogenous gasotransmitter, besides nitric oxide (NO) and carbon monoxide (CO) [12]. Several studies have shown that it plays a role in different physiopathological processes; for example, it acts as cardioprotective agent [13], modulates inflammation [14], reduces oxidative stress [15], induces bronchial relaxation [16] and provides a cytoprotective effect [17].
Despite the interesting properties of H 2 S in the human body, as a gaseous compound, it cannot be considered an ideal drug. For this reason, scientists worked on the development of molecules able to release endogenously H 2 S (named H 2 S donors), that could be used as biological instruments and potential drugs [13]. The most promising H 2 S-releasing compounds are the synthetic donors, characterized by an enhanced safety profile and a better pharmacokinetic profile that mimic the time course of the physiologic H 2 S release.
The discovery of the enzymes that mediate H 2 S production in ocular tissues suggested a potential physiological role for this gasotransmitter in the eye [18]. Different ocular diseases related to retinal degeneration like glaucoma, AMD (age-related macular degeneration) and DR (diabetic retinopathy) are characterized by the reduction of endogenous H 2 S levels and expression of H 2 S synthetizing enzymes [19]. Several studies have shown that exogenous H 2 S released by molecular donors can reduce RGCs' damage related to oxidative stress and elevated hydrostatic pressure [15,[20][21][22][23]. The vasorelaxant effect associated with H 2 S has also been widely demonstrated in ocular vasculature, improving blood flow in the eye [15,21]. In addition, H 2 S plays a role in ocular structures implicated in AH production and outflow as well as in IOP control [18].
In the last decades, H 2 S-releasing molecules have been linked to several pharmaceutical active compounds to synthetize novel molecular hybrids with the purpose of associating the functionality of the parent drugs and endogenous H 2 S. An interesting example of an antiglaucoma drug conjugated to a H 2 S donor is ACS67, a molecular hybrid of latanoprost acid and ADT-OH, a derivative of anethole dithiolethione. Studies confirmed the potentiality of this drug that combines the IOP-lowering effect of latanoprost and the neuroprotective activity of H 2 S, released by ADT-OH [24].
On the basis of the data reported above, and considering the expertise of our research group in the field of H 2 S donors and their applications to synthetize novel chemical entities [25][26][27][28], in this experimental work we designed, synthetized and characterized new molecular hybrids between drugs for the treatment of glaucoma and H 2 S-donating moieties. The aim was to synthesize a compound which combines the action of antiglaucomatous drugs and H 2 S released by donors. The idea was to enhance the efficacy of the IOPlowering medications with the promising effect of H 2 S to provide a heightened therapy. The molecular hybrids must be stable enough to be administered, but once absorbed in the eye they undergo in vivo metabolic reactions that trigger the disintegration of the hybrids, allowing the antiglaucoma drug and the H 2 S donor to interact with their biological targets. By the application of these new entities, we expect a reduction in the administered dosage and side effects.

Brinzolamide Derivatives
brids, allowing the antiglaucoma drug and the H2S donor to interact with their biological targets. By the application of these new entities, we expect a reduction in the administered dosage and side effects.

Chemistry
Chemical structures of compounds 1a-1d, 2a-2d and 3a-3d are represented in Table  1. The synthetic routes for the synthesis of molecular hybrids of brinzolamide (1a-1d), betaxolol (2a-2d) and brimonidine (3a-3d) are summarized, respectively, in Schemes 1-3. Table 1. Chemical structures of new molecular hybrids between antiglaucoma drugs and H2S donors. Values of Cmax (μM) relative to H2S generation following the incubation in the assay buffer of the free H2S donors (7-10) and antiglaucoma hybrids (1a-1d, 2a-2d and 3a-3d) at concentration of 100 μM, in the presence (+ L-Cys) and in the absence (-L-Cys) of L-cysteine 4 mM; n.d. = not detected (H2S release < 0.4 μM). Data are reported as means ± SEM. brids, allowing the antiglaucoma drug and the H2S donor to interact with their biological targets. By the application of these new entities, we expect a reduction in the administered dosage and side effects.

Chemistry
Chemical structures of compounds 1a-1d, 2a-2d and 3a-3d are represented in Table  1. The synthetic routes for the synthesis of molecular hybrids of brinzolamide (1a-1d), betaxolol (2a-2d) and brimonidine (3a-3d) are summarized, respectively, in Schemes 1-3. Table 1. Chemical structures of new molecular hybrids between antiglaucoma drugs and H2S donors. Values of Cmax (μM) relative to H2S generation following the incubation in the assay buffer of the free H2S donors (7-10) and antiglaucoma hybrids (1a-1d, 2a-2d and 3a-3d) at concentration of 100 μM, in the presence (+ L-Cys) and in the absence (-L-Cys) of L-cysteine 4 mM; n.d. = not detected (H2S release < 0.4 μM). Data are reported as means ± SEM. brids, allowing the antiglaucoma drug and the H2S donor to interact with their biological targets. By the application of these new entities, we expect a reduction in the administered dosage and side effects.

Chemistry
Chemical structures of compounds 1a-1d, 2a-2d and 3a-3d are represented in Table  1. The synthetic routes for the synthesis of molecular hybrids of brinzolamide (1a-1d), betaxolol (2a-2d) and brimonidine (3a-3d) are summarized, respectively, in Schemes 1-3. Table 1. Chemical structures of new molecular hybrids between antiglaucoma drugs and H2S donors. Values of Cmax (μM) relative to H2S generation following the incubation in the assay buffer of the free H2S donors (7-10) and antiglaucoma hybrids (1a-1d, 2a-2d and 3a-3d) at concentration of 100 μM, in the presence (+ L-Cys) and in the absence (-L-Cys) of L-cysteine 4 mM; n.d. = not detected (H2S release < 0.4 μM). Data are reported as means ± SEM. targets. By the application of these new entities, we expect a reduction in the administered dosage and side effects.

Brinzolamide Derivatives
targets. By the application of these new entities, we expect a reduction in the administered dosage and side effects.
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H2S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H2S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H2S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H2S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H2S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H2S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol 0.9 ± 0.4 n.d.
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H2S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H2S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H2S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H2S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol 6.2 ± 0.5 0.8 ± 0.1
The synthesis of compound 1d started from the conversion of brinzolamide in its succinic derivative 4 by treatment with succinic anhydride in acetonitrile. The following coupling reaction of intermediate 4, solubilized in DMF, with HPI 10 was performed using TBTU, HOBt and N,N-diisopropylethylamine as coupling agents.
Unlike compounds 2a-2c, for compound 2d, the spacer-H 2 S donor moiety is linked to the alcoholic function of betaxolol by an ester bond, instead of to the aminic group, due to the unsuccessful reaction between HPI and tert-butyl bromoacetate. In this case, betaxolol 2 was treated with an excess of succinic anhydride and a catalytic amount of DMAP in anhydrous CH 2 Cl 2 to produce the corresponding hemisuccinated ester that was linked to HPI 10 in the presence of EDAC·HCl and DMAP, obtaining the compound 2d.
Via one-pot reaction, brimonidine 3 solubilized in anhydrous DMF was first converted into its derivative 5 by treatment with succinic anhydride and DMAP, and then the obtained intermediate was linked to the H 2 S donors (7-10) by means of EDAC·HCl and DMAP, producing the corresponding compounds 3a-3d.
The H 2 S-releasing moieties 7 and 10 were commercially available. ADT-OH 8 was synthetized by reacting trans-anethole and sulfur in DMF according to a process reported in the literature [31]. The H 2 S donor HBTA 9 was obtained following the synthetic procedure described by our research group [27]. Scheme 4 reports the synthetic route for introducing an acetic spacer on the H 2 S donors 7-9. TBZ, ADT-OH and HBTA were reacted with tert-butyl bromoacetate in the presence of NaH in DMF to produce intermediates 7a-9a, which were successfully deprotected by treatment with a 10% (v/v) TFA solution in CH 2 Cl 2 affording the desired intermediates 7b-9b.
anhydrous CH2Cl2 to produce the corresponding hemisuccinated ester that was linked to HPI 10 in the presence of EDAC·HCl and DMAP, obtaining the compound 2d.
Via one-pot reaction, brimonidine 3 solubilized in anhydrous DMF was first converted into its derivative 5 by treatment with succinic anhydride and DMAP, and then the obtained intermediate was linked to the H2S donors (7-10) by means of EDAC·HCl and DMAP, producing the corresponding compounds 3a-3d.
The H2S-releasing moieties 7 and 10 were commercially available. ADT-OH 8 was synthetized by reacting trans-anethole and sulfur in DMF according to a process reported in the literature [31]. The H2S donor HBTA 9 was obtained following the synthetic procedure described by our research group [27]. Scheme 4 reports the synthetic route for introducing an acetic spacer on the H2S donors 7-9. TBZ, ADT-OH and HBTA were reacted with tert-butyl bromoacetate in the presence of NaH in DMF to produce intermediates 7a-9a, which were successfully deprotected by treatment with a 10% (v/v) TFA solution in CH2Cl2 affording the desired intermediates 7b-9b.

Amperometric Evaluation of H2S Release
The H2S-generating properties of the compounds 1a-1d, 2a-2d and 3a-3d were evaluated by amperometry, allowing a "real time" detection of the released H2S with high sensitivity and selectivity [32]. The assay was performed in an aqueous phosphate buffer, in the absence or in the presence of L-cysteine, whose thiol group mimics the endogenous free thiols in the cells. In Table 1, the Cmax values are reported, representing the highest concentration of H2S (μM) recorded during the experiments and released by the H2S donating moieties and molecular hybrids (100 μM) in the experimental conditions.
As illustrated in Figure 1, the amperometric assay demonstrated that in the absence of L-cysteine, all the compounds had a completely negligible release of H2S (<0,4 μM),

Amperometric Evaluation of H 2 S Release
The H 2 S-generating properties of the compounds 1a-1d, 2a-2d and 3a-3d were evaluated by amperometry, allowing a "real time" detection of the released H 2 S with high sensitivity and selectivity [32]. The assay was performed in an aqueous phosphate buffer, in the absence or in the presence of L-cysteine, whose thiol group mimics the endogenous free thiols in the cells. In Table 1, the C max values are reported, representing the highest concentration of H 2 S (µM) recorded during the experiments and released by the H 2 S donating moieties and molecular hybrids (100 µM) in the experimental conditions.
As illustrated in Figure 1, the amperometric assay demonstrated that in the absence of L-cysteine, all the compounds had a completely negligible release of H 2 S (<0,4 µM), except ADT-OH 8 and HPI 10. These data proved that the presence of a thiol group activates and/or enhances the H 2 S generation from the tested compounds. Therefore, they act as "smart H 2 S donors" since these agents can donate the gaseous transmitter only in a biological environment, i.e., in the presence of organic thiols [26,32,33]. Otherwise, ADT-OH 8 and HPI 10 were able to release H 2 S both in the absence and in the presence of L-cysteine, due to their susceptibility to both a hydrolytic and thiol-dependent mechanism of release.
All brinzolamide hybrids (compounds 1a-1d) showed an L-cysteine-dependent generation of H 2 S. Nevertheless, the hybrid brinzolamide-TBZ (1a) had the lowest release (C max = 0.4 ± 0.2 µM) while the compound 1c (brinzolamide-HBTA) showed a slow and considerable production of H 2 S and within the series of the brinzolamide hybrids, demonstrated the highest C max (3.5 ± 0.3 µM). Amperometric data obtained from 1c confirmed the promising results collected by our research group [27], suggesting HBTA as an innovative and effective thiol-triggered H 2 S donor.
The molecular hybrids synthetized, starting from betaxolol (2a-2d), had a weak H 2 S release, enhanced by the presence of L-cysteine. The curves for H 2 S release vs. time in the absence or in the presence of L-Cys for compound 2a (betaxolol-TBZ) were almost overlapping.
Compounds 3a-3d required the presence of L-Cys to obtain a detectable generation of H 2 S. The hybrid brimonidine-HPI 3d showed the best releasing profile, with progressive and time-related slow gas donation. The compound 3d produced a significant H 2 S generation with a C max value of 6.2 ± 0.5 µM. In addition, in this case, data from amperometric assay corroborated the studies indicating that isothiocyanates are promising H 2 S donors [32,33]. Furthermore, as illustrated by Lin et al., the endogenous H 2 S release from isothiocyanates occurs in the presence of thiols (mainly GSH or L-Cys). In particular, the authors showed that isothiocyanates react rapidly with the L-Cys to form an adduct, which then undergoes an intramolecular cyclization reaction to finally release H 2 S [34]. In addition, the electronic effect of the substituents linked to the isothiocyanate may influence the H 2 S formation rate. H2S [34]. In addition, the electronic effect of the substituents linked to the isothiocyanate may influence the H2S formation rate.

Intracellular H2S Release in HCEs
The H2S-releasing properties of the novel molecular hybrids were also tested in human primary corneal epithelial cells (HCEs) to verify the H2S formation into the cellular environment without adding any exogenous thiol. This method allows one to understand

Intracellular H 2 S Release in HCEs
The H 2 S-releasing properties of the novel molecular hybrids were also tested in human primary corneal epithelial cells (HCEs) to verify the H 2 S formation into the cellular environment without adding any exogenous thiol. This method allows one to understand the behavior of the H 2 S donors in the presence of a physiological level of intracellular L-cysteine, since we use non-immortalized corneal cells.
The detection of intracellular H 2 S was performed by spectrofluorometric measurements using the dye 3 -methoxy-3-oxo-3H-spiro-(isobenzofuran-1,9 -xanthen)-6 -yl-(pyridin-2-yldisulfanyl) benzoate (Washington State Probe-1, WSP-1). WSP-1 can react specifically and irreversibly with H 2 S generated by the tested compounds, releasing a fluorophore detectable with a spectrofluorometer. The increase in the fluorescence compared to the blank is expressed as fluorescence index (FI) [33]. The FI values of the H 2 S donors and the hybrids were compared to the FI value of diallyl disulfide (DADS), considered as reference sulfide donor and responsible for significant H 2 S production (p < 0.001). The addition of the vehicle (1% DMSO) in the experimental conditions reflects the endogenous production of H 2 S in the cells.
The experiments were performed in HCEs because the cornea is the major route for topical ocular drug absorption and the corneal epithelium is the most anterior layer of the cornea as well as the main barrier for drug absorption from the tear fluid to the anterior chamber of the eye [35,36].
In Figure 2, H 2 S formation values of the donating moieties (100 µM) 7-10 are represented. The compounds TBZ 7 and ADT-OH 8 incubated in HCEs led to a weak and not significant H 2 S-release, almost comparable to that of the vehicle, showing their inability to enter the cell and produce H 2 S. On the other hand, HBTA 9 and HPI 10 promoted an elevated and significant (p < 0.001) increase of WSP-1 fluorescence, comparable to the reference H 2 S donor DADS. The graphs reporting the histograms of the intracellular H 2 S release after the incubation of the compounds are subjected to area-under-the-curve analysis of the fluorescence increase monitored for 50 min (For a better characterization and for a better comprehension of the results, see the graphs of the kinetic included in the Supplementary Data). the behavior of the H2S donors in the presence of a physiological level of intracellular Lcysteine, since we use non-immortalized corneal cells. The detection of intracellular H2S was performed by spectrofluorometric measurements using the dye 3′-methoxy-3-oxo-3H-spiro-(isobenzofuran-1,9′-xanthen)-6′yl-(pyridin-2-yldisulfanyl) benzoate (Washington State Probe-1, WSP-1). WSP-1 can react specifically and irreversibly with H2S generated by the tested compounds, releasing a fluorophore detectable with a spectrofluorometer. The increase in the fluorescence compared to the blank is expressed as fluorescence index (FI) [33]. The FI values of the H2S donors and the hybrids were compared to the FI value of diallyl disulfide (DADS), considered as reference sulfide donor and responsible for significant H2S production (p < 0.001). The addition of the vehicle (1% DMSO) in the experimental conditions reflects the endogenous production of H2S in the cells.
The experiments were performed in HCEs because the cornea is the major route for topical ocular drug absorption and the corneal epithelium is the most anterior layer of the cornea as well as the main barrier for drug absorption from the tear fluid to the anterior chamber of the eye [35,36].
In Figure 2, H2S formation values of the donating moieties (100 μM) 7-10 are represented. The compounds TBZ 7 and ADT-OH 8 incubated in HCEs led to a weak and not significant H2S-release, almost comparable to that of the vehicle, showing their inability to enter the cell and produce H2S. On the other hand, HBTA 9 and HPI 10 promoted an elevated and significant (p < 0.001) increase of WSP-1 fluorescence, comparable to the reference H2S donor DADS. The graphs reporting the histograms of the intracellular H2S release after the incubation of the compounds are subjected to areaunder-the-curve analysis of the fluorescence increase monitored for 50 min (For a better characterization and for a better comprehension of the results, see the graphs of the kinetic included in the Supplementary Data).

Figure 2.
Cumulative H2S release (expressed as area under the curve of the WSP-1 fluorescence in the recording time) after the incubation of the vehicle, the tested compounds (7-10) and diallyl disulfide (DADS) (100 μM). Data were expressed as mean ± standard error. Three different experiments were carried out, each in triplicate. ANOVA and Student's t-test were applied as statistical analyses; when required, the Bonferroni post hoc test was used to calculate the significance level (*** p < 0.001).

Figure 2.
Cumulative H 2 S release (expressed as area under the curve of the WSP-1 fluorescence in the recording time) after the incubation of the vehicle, the tested compounds (7-10) and diallyl disulfide (DADS) (100 µM). Data were expressed as mean ± standard error. Three different experiments were carried out, each in triplicate. ANOVA and Student's t-test were applied as statistical analyses; when required, the Bonferroni post hoc test was used to calculate the significance level (*** p < 0.001).
The intracellular H 2 S-releasing profiles of compounds 1a-1d (100 µM), reported in Figure 3, show that all brinzolamide hybrids evocated a significant H 2 S release. Interestingly, compound 1a (brinzolamide-TBZ) had an enhanced H 2 S production compared to the TBZ-free moiety. The addition of the hybrid brinzolamide-ADTOH (1b) in HCEs caused a higher increase in FI value than ADT-OH by itself. The incubation of compounds 1c and 1d (brinzolamide-HBTA and brinzolamide-HPI, respectively) promoted a significant intracellular H 2 S release (p < 0.001). The intracellular H2S-releasing profiles of compounds 1a-1d (100 μM), reported in Figure 3, show that all brinzolamide hybrids evocated a significant H2S release. Interestingly, compound 1a (brinzolamide-TBZ) had an enhanced H2S production compared to the TBZ-free moiety. The addition of the hybrid brinzolamide-ADTOH (1b) in HCEs caused a higher increase in FI value than ADT-OH by itself. The incubation of compounds 1c and 1d (brinzolamide-HBTA and brinzolamide-HPI, respectively) promoted a significant intracellular H2S release (p < 0.001).  (1a-1d), the native H2S donors and diallyl disulfide (DADS) (100 μM). Data were expressed as mean ± standard error. Three different experiments were carried out, each in triplicate. ANOVA and Student's t-test were applied as statistical analyses; when required, the Bonferroni post hoc test was used to calculate the significance level (* p < 0.05; *** p < 0.001).
Compounds 2a betaxolol-TBZ and 2b betaxolol-ADTOH did not cause any significant increase in fluorescence (Figure 4). The addition of molecular hybrids betaxolol-HBTA (2c) and betaxolol-HPI (2d) to WSP-1-preloaded HCEs evoked a mild but significant increase in the intracellular H2S levels (p < 0.001).  Three different experiments were carried out, each in triplicate. ANOVA and Student's t-test were applied as statistical analyses; when required, the Bonferroni post hoc test was used to calculate the significance level (* p < 0.05; *** p < 0.001).
Compounds 2a betaxolol-TBZ and 2b betaxolol-ADTOH did not cause any significant increase in fluorescence ( Figure 4). The addition of molecular hybrids betaxolol-HBTA (2c) and betaxolol-HPI (2d) to WSP-1-preloaded HCEs evoked a mild but significant increase in the intracellular H 2 S levels (p < 0.001). The intracellular H2S-releasing profiles of compounds 1a-1d (100 μM), reported in Figure 3, show that all brinzolamide hybrids evocated a significant H2S release. Interestingly, compound 1a (brinzolamide-TBZ) had an enhanced H2S production compared to the TBZ-free moiety. The addition of the hybrid brinzolamide-ADTOH (1b) in HCEs caused a higher increase in FI value than ADT-OH by itself. The incubation of compounds 1c and 1d (brinzolamide-HBTA and brinzolamide-HPI, respectively) promoted a significant intracellular H2S release (p < 0.001). Compounds 2a betaxolol-TBZ and 2b betaxolol-ADTOH did not cause any significant increase in fluorescence ( Figure 4). The addition of molecular hybrids betaxolol-HBTA (2c) and betaxolol-HPI (2d) to WSP-1-preloaded HCEs evoked a mild but significant increase in the intracellular H2S levels (p < 0.001).  Three different experiments were carried out, each in triplicate. ANOVA and Student's t-test were applied as statistical analyses; when required, the Bonferroni post hoc test was used to calculate the significance level (*** p < 0.001).
In Figure 5, the results of the fluorometric assay of brimonidine hybrids are graphically represented. All the compounds led to a significant release of hydrogen sulfide (p < 0.001), except for the ADT-OH conjugated hybrid (3b). Among the compounds 3a-3d, the molecular hybrid 3d (brimonidine-HPI) showed the highest increase in fluorescence. In Figure 5, the results of the fluorometric assay of brimonidine hybrids are graphically represented. All the compounds led to a significant release of hydrogen sulfide (p < 0.001), except for the ADT-OH conjugated hybrid (3b). Among the compounds 3a-3d, the molecular hybrid 3d (brimonidine-HPI) showed the highest increase in fluorescence. Analyzing the data from the amperometric and the fluorometric assays, the molecular hybrids synthetized by coupling HBTA 9 and HPI 10 with antiglaucoma drugs (1-3) released a higher amount of H2S in aqueous buffer as well as in the cells, compared to the molecular hybrids of TBZ 7 and ADT-OH 8. Furthermore, evaluating the influence of the antiglaucoma drugs in the release of H2S, betaxolol hybrids demonstrated a weak generation of sulfide when compared to brinzolamide and brimonidine derivatives.
Therefore, compounds 1c, 1d and 3d showed the best releasing profiles, leading to an enhanced H2S production. Besides the amount of the gasotransmitter produced, the H2S-releasing kinetic also influences biological activity. The amperometic assay demonstrated that these hybrids had a progressive and long-lasting release of H2S in the presence of L-cysteine, acting as smart donors. These features are considered as indispensable for the potential clinical application of H2S donors, since they avoid the side effects related to a fast release (typical of the sulfide and hydrosulfide salts) and also mimic the endogenous H2S production. Three different experiments were carried out, each in triplicate. ANOVA and Student's t-test were applied as statistical analyses; when required, the Bonferroni post hoc test was used to calculate the significance level (*** p < 0.001).
Analyzing the data from the amperometric and the fluorometric assays, the molecular hybrids synthetized by coupling HBTA 9 and HPI 10 with antiglaucoma drugs (1-3) released a higher amount of H 2 S in aqueous buffer as well as in the cells, compared to the molecular hybrids of TBZ 7 and ADT-OH 8. Furthermore, evaluating the influence of the antiglaucoma drugs in the release of H 2 S, betaxolol hybrids demonstrated a weak generation of sulfide when compared to brinzolamide and brimonidine derivatives.
Therefore, compounds 1c, 1d and 3d showed the best releasing profiles, leading to an enhanced H 2 S production. Besides the amount of the gasotransmitter produced, the H 2 Sreleasing kinetic also influences biological activity. The amperometic assay demonstrated that these hybrids had a progressive and long-lasting release of H 2 S in the presence of L-cysteine, acting as smart donors. These features are considered as indispensable for the potential clinical application of H 2 S donors, since they avoid the side effects related to a fast release (typical of the sulfide and hydrosulfide salts) and also mimic the endogenous H 2 S production.

Materials and Methods
Brinzolamide and brimonidine were purchased from Abcr (Karlsruhe, Germany); betaxolol was purchased from Carbosynth (Compton, UK). All reagents, solvents and other chemicals were commercial products obtained from Merck (Darmstadt, Germany). Melting points, determined using a Buchi Melting Point B-540 instrument (Flawil, Switzerland), are uncorrected and represent values obtained on recrystallized or chromatographically purified material. Spectra of 1 H and 13 C NMR were recorded on a Bruker Advanced 400 MHz spectrometer (Billerica, MA, USA). Spectra of brinzolamide and brimonidine derivatives were recorded in DMSO-d 6 . Spectra of betaxolol hybrids were recorded in CD 3 OD and CDCl 3 (compound 2d). Chemical shifts are reported in ppm. The following abbreviations are used to describe peak patterns when appropriate: s (singlet), d (doublet), t (triplet), m (multiplet), q (quartet), qt (quintet), dd (doublet of doublet), td (triplet of doublets), bs (broad singlet). Mass spectra of the intermediates and final products were recorded on an LTQ-XL mass spectrometer equipped with a HESI ion source (Thermo Fisher Scientific, Waltham, MA, USA). All reactions were followed by thin-layer chromatography, carried out on Merck silica gel 60 F 254 plates with a fluorescent indicator, and the plates were visualized with UV light (254 nm). Preparative chromatographic purifications were performed using a silica gel column (Kieselgel 60). Solutions were concentrated with a Buchi R-114 rotary evaporator at low pressure.   Following the synthetic procedure described above for 1a, compound 1b was synthetized starting from brinzolamide 1 (1.00 g; 2.61 mmol) and the derivative 8b (0.742 g; 2.61 mmol), and isolated as an orange solid. Yield: 0.889 g; 52.4%. Mp: 154.1-155.6 • C. 1  The synthesis of compound 1d occurs in two steps. The first reaction was performed in acetonitrile (20 mL) as solvent, with azeotropic elimination of water from the system [37]. Succinic anhydride (0.287 g; 2.87 mmol) was added to a solution of brinzolamide 1 (1.00 g; 2.61 mmol) and the mixture was stirred overnight at reflux. The solvent was evaporated under reduced pressure and the crude residue was then purified by silica gel open chromatography (dichloromethane/methanol 9:1 v/v) to obtain the acid derivative 4 as colorless oil. Yield: 0.891 g; 70. 6%  The analysis of 1 H, 13 C and bidimensional NMR spectra showed that betaxolol hybrids 2a-2c are a mixture of cis/trans isomers ( Figure 6). As reported in details in the literature, unsymmetrically N,N-disubstituted amides are characterized by a hindered rotation around the C(O)-N bond but the energy difference between the two conformations is small and the molecules are a combination of cis/trans isomers [38][39][40][41]. The rate of conversion between conformational isomers of betaxolol hybrids is sufficiently slow to allow a chemical shift difference of signals arising from cis and trans isomers.

Statistical Analysis
Experimental data were analyzed by a computer fitting procedure (software: Graph-Pad Prism 6.0) and expressed as mean ± standard error; three different experiments were performed, each carried out in three replicates. ANOVA and Student's t-test were selected as statistical analyses; when required, the Bonferroni post hoc test was used. p < 0.05 was considered as representative of significant statistical differences.

Conclusions
For most forms of glaucoma, including normotensive glaucoma, pharmacological treatment is currently based on IOP control through topical medications. However, the last topical agent for glaucoma therapy approved by the Food and Drug Administration (FDA) dates back to more than 20 years ago [43]. Therefore, with the increasing prevalence of glaucoma worldwide, the exigency of new therapies is emerging.
In this work, we synthetized and characterized new molecular hybrids between currently available drugs for glaucoma therapy and H 2 S-releasing compounds to improve the efficacy of antiglaucoma medications and reduce side effects.
The new molecular entities were tested for their H 2 S-releasing properties via amperometric and fluorometric assays.
In the amperometric studies, all the synthetized hybrids showed a completely negligible H 2 S production in the absence of L-Cys, proving that the thiol group acts as a trigger for the release of the sulfide. Betaxolol hybrids (compounds 2a-2d) demonstrated poor H 2 S-releasing properties even in the presence of L-Cys. This behaviour was also confirmed in the fluorometric assay.
Even if H 2 S reaches low micromolar levels, it is characterized by a hormetic behavior: high concentrations of H 2 S are toxic and H 2 S donors able to donate high amount of H 2 S showed antitumoral activity [27,44]. To obtain benefits from H 2 S donation, the amount of H 2 S should be at low micromolar level, mimicking its physiological production. This low concentration has been demonstrated to activate, for example, Nrf2, to inhibit Nf-kb, and to protect endothelium from harmful stimuli [45][46][47]. Additionally, as reported in the literature [48], the therapeutic concentration range of H 2 S in the ocular tissues is 100 nM-100 µM.
These preliminary results confirm hybridization as a promising strategy in the drug design process. By the synthesis of a new molecular entity through the combination of two or more identical or different drugs, with or without a linker, the aim is to enhance the efficacy of the parent agents [49].
Moreover, based on these results, the idea of combining powerful H 2 S donors such as HBTA or HPI with efficacious IOP-lowering drugs such as prostaglandin analogs could be an interesting, novel perspective to obtain novel antiglaucoma drugs.