PhSeZnCl in the Synthesis of Steroidal β-Hydroxy-Phenylselenides Having Antibacterial Activity

We report here the reaction of in situ prepared PhSeZnCl with steroid derivatives having an epoxide as an electrophilic functionalization. The corresponding ring-opening reaction resulted to be regio- and stereoselective affording to novel phenylselenium-substituted steroids. Assessment of their antibacterial properties against multidrug-resistant bacteria, such as Pseudomonas aeruginosa Xen 5 strain, indicates an interesting bactericidal activity and their ability to prevent bacterial biofilm formation.


Introduction
The last few decades have seen a growing interest in the synthesis of organoselenium compounds due to some promising biological activities, reported in the recent literature [1][2][3]. Selenium is present in mammalian organisms in the form of selenoenzymes, embedded in the proteinogenic amino acid selenocysteine. All the selenoproteins demonstrated redox properties and have a crucial role in the redox modulation as well as in the control of the redox homeostasis of the living systems, playing also a role in the cell's protection against the oxidative stress [4]. The prooxidant activity of organoselenium compounds was recently described as an interesting property to be directed toward specific targets developing antibacterial [5], antiviral [6], and antifungal agents [7], hormetines [8], and enzyme inhibitors [9]. Furthermore, direct and indirect interferences with the redox homeostasis and the redox signaling can produce an anticancer effect affecting the differentiation, proliferation, senescence and death pathways in the cells [10].
Even if many physiological and pathological mechanisms involving organoselenium derivatives need to be clarified, the recent introduction of Ebselen ® and Ebselen-like compounds in clinical trials for the treatment of diabetes complications and non-small cell lung cancer, further demonstrate the current interest in study these derivatives for medical purposes [11,12].
Till now, few examples of selenosteroids were reported in the literature and they were tested and proved to be interesting antiproliferative agents following a prooxidant mechanism.
Some representative examples are reported in Figure 1 [13][14][15], selenium can be embedded as selenourea, selenocyanate or alkyl/aryl selenide directly introduced in C-3 or C-6 through the S N 2 reaction of a selenolates with a tosylate (VI) or an epoxide (V) affording, in this letter case to a β-hydroxyselenide [16,17]. Int selenolates with a tosylate (VI) or an epoxide (V) affording, in this letter case to a β-hydroxyselenide [16,17]. During the last ten years, some of us deeply investigated the use of nucleophilic selenium reagents, in the form of zinc selenolates, for the functionalization of electrophilic organic substrates [18]. Among these reagents the PhSeZnCl, easily prepared through the oxidative insertion of elemental zinc into Se-halogen bound of PhSeCl, it was the first bench stable organic selenolate [19], it is nowadays commercially available, and someone recently named as Santi's reagent [20]. These reagents demonstrate a broad range of applicability showing in several cases a strong rate acceleration when the reaction is performed in "on water" conditions [21][22][23].
In addition, its use was recently described also in the ring opening reactions of aziridines [24] and epoxides for the optimization of a total synthesis [25] as well as in the functionalization of preformed polymers [26], demonstrating the effective applicability of the method also starting from polyfunctionalized substrates. The ring opening of epoxide with PhSeZnCl is generally highly regioselective and the presence of the zinc as Lewis acid was demonstrated to be in some cases important for the regioselective control of the reaction [19].

Chemistry
In the present work, we explored the use of PhSeZnCl for the functionalization of different steroid derivatives having an epoxide as an electrophilic reactive center, in order to obtain new molecular selenosteroids as prototypes for the investigation of the antibacterial activity. The optimization of the reaction conditions was performed starting from the epoxide 1 as a 4:1 mixture of the α and β isomer, respectively.
The reaction of 5,6-epoxycholestane 1 with solid PhSeZnCl was firstly investigated using the conditions reported for the opening of epoxide both in water suspension and THF solution at room temperature [19] but these procedures did not afford the desired product. Differently, when the reagent was prepared in situ in THF and the epoxide 1 was refluxed for 3 h with the reagent the βhydroxyselenide 2 was obtained in 60% yield (75% on converted material), as depicted in Scheme 1. During the last ten years, some of us deeply investigated the use of nucleophilic selenium reagents, in the form of zinc selenolates, for the functionalization of electrophilic organic substrates [18]. Among these reagents the PhSeZnCl, easily prepared through the oxidative insertion of elemental zinc into Se-halogen bound of PhSeCl, it was the first bench stable organic selenolate [19], it is nowadays commercially available, and someone recently named as Santi's reagent [20].
These reagents demonstrate a broad range of applicability showing in several cases a strong rate acceleration when the reaction is performed in "on water" conditions [21][22][23].
In addition, its use was recently described also in the ring opening reactions of aziridines [24] and epoxides for the optimization of a total synthesis [25] as well as in the functionalization of preformed polymers [26], demonstrating the effective applicability of the method also starting from polyfunctionalized substrates. The ring opening of epoxide with PhSeZnCl is generally highly regioselective and the presence of the zinc as Lewis acid was demonstrated to be in some cases important for the regioselective control of the reaction [19].

Chemistry
In the present work, we explored the use of PhSeZnCl for the functionalization of different steroid derivatives having an epoxide as an electrophilic reactive center, in order to obtain new molecular selenosteroids as prototypes for the investigation of the antibacterial activity. The optimization of the reaction conditions was performed starting from the epoxide 1 as a 4:1 mixture of the α and β isomer, respectively.
The reaction of 5,6-epoxycholestane 1 with solid PhSeZnCl was firstly investigated using the conditions reported for the opening of epoxide both in water suspension and THF solution at room temperature [19] but these procedures did not afford the desired product. Differently, when the reagent was prepared in situ in THF and the epoxide 1 was refluxed for 3 h with the reagent the β-hydroxyselenide 2 was obtained in 60% yield (75% on converted material), as depicted in Scheme 1. The nucleophilic attack to the epoxide ring occurs exclusively on the less sterically hindered carbon (C6) due to the presence of axial C-19 methyl group, indicating that an S N 2 mechanism is involved in the process and affording the corresponding trans-hydroxy selenides 2. Only the trans hydroxyl selenide 2 was observed and the unreacted 5β,6β-epoxide was quantitatively recovered after chromatographic purification. The reason of the non-reactivity of 5β,6β-epoxide with nucleophilic reagents was recently explained and deal with the unfavorable formation of a constrained structure that should arise from the trans-diaxial opening at C6 with the hydroxyl at C5 in the syn position with regard to the C19 [27]. A separate experiment with epoxycholestane 1 and (PhSe) 2 in the presence of NaBH 4 was performed. After 3 h, TLC monitoring showed the consumption of a part of the starting material 1. On the basis of 1 H NMR spectral analysis, it was established that only the α-epoxide 1 reacted while the β-isomer of 1 remained intact. Moreover, after 3 h of the reaction, 3β-hydroxy-5α,6α-epoxycholestane (20%) was still present in the reaction mixture.
Compound 2 was fully characterized by NMR, IR and MS spectroscopies and the collected data correspond to those previously reported by Rodrigues et al. [16].
In the optimized protocol, the zinc (1.0 equivalent) activated by treatment with HCl 10%, was added to the solution of PhSeCl (1.0 equivalent) in anhydrous THF and heated at the reflux temperature till the discoloration of the solution that is assumed to be indicative for the formation in situ of PhSeZnCl. At this point, the substrate 1 (1.0 equiv) dissolved in THF was added. The reaction was refluxed for an additional 3 h and monitored by thin-layer chromatography (TLC). The nucleophilic attack to the epoxide ring occurs exclusively on the less sterically hindered carbon (C6) due to the presence of axial C-19 methyl group, indicating that an SN2 mechanism is involved in the process and affording the corresponding trans-hydroxy selenides 2. Only the trans hydroxyl selenide 2 was observed and the unreacted 5β,6β-epoxide was quantitatively recovered after chromatographic purification. The reason of the non-reactivity of 5β,6β-epoxide with nucleophilic reagents was recently explained and deal with the unfavorable formation of a constrained structure that should arise from the trans-diaxial opening at C6 with the hydroxyl at C5 in the syn position with regard to the C19 [27]. A separate experiment with epoxycholestane 1 and (PhSe)2 in the presence of NaBH4 was performed. After 3 h, TLC monitoring showed the consumption of a part of the starting material 1. On the basis of 1 H NMR spectral analysis, it was established that only the α-epoxide 1 reacted while the β-isomer of 1 remained intact. Moreover, after 3 h of the reaction, 3β-hydroxy-5α,6α-epoxycholestane (20%) was still present in the reaction mixture. Compound 2 was fully characterized by NMR, IR and MS spectroscopies and the collected data correspond to those previously reported by Rodrigues et al. [16].
In the optimized protocol, the zinc (1.0 equivalent) activated by treatment with HCl 10%, was added to the solution of PhSeCl (1.0 equivalent) in anhydrous THF and heated at the reflux temperature till the discoloration of the solution that is assumed to be indicative for the formation in situ of PhSeZnCl. At this point, the substrate 1 (1.0 equiv) dissolved in THF was added. The reaction was refluxed for an additional 3 h and monitored by thin-layer chromatography (TLC).
Using the above-described conditions, the scope of epoxy substrates 1, 3, 5, 6 and 8 was explored. The results are collected in Table 1. Interestingly the mild conditions of the procedure resulted compatible with the sensitive spiroacetal system [28] in 3 and the sterically hindered epoxide 5 [29] was totally unreactive. The reactions of 1,2-epoxysteroids 6 and 8 with the in situ generated organoselenium reagent gave 1β,3β-dihydroxy-2β-phenylselenylcholestane (7) and 2β,3αdihydroxy-1α-phenylselenylcholestane (9), respectively. For the accurate determination of the stereochemistry of the centers formed in the C-1 and C-2 positions, two-dimensional NMR experiments were performed (COrrelation SpectrocpY (COSY), heteronuclear single quantum Using the above-described conditions, the scope of epoxy substrates 1, 3, 5, 6 and 8 was explored. The results are collected in Table 1. Interestingly the mild conditions of the procedure resulted compatible with the sensitive spiroacetal system [28] in 3 and the sterically hindered epoxide 5 [29] was totally unreactive. The reactions of 1,2-epoxysteroids 6 and 8 with the in situ generated organoselenium reagent gave 1β,3β-dihydroxy-2β-phenylselenylcholestane (7) and 2β,3α-dihydroxy-1α-phenylselenylcholestane (9), respectively. For the accurate determination of the stereochemistry of the centers formed in the C-1 and C-2 positions, two-dimensional NMR experiments were performed (COrrelation SpectrocpY (COSY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC)) in 400 MHz instrument. For compound 7, the coupling system inferred from the correlation spectra indicates that phenylselenyl group is bonded to C-2, since 2-H is coupled to both adjacent 1-H and 3-H. In addition, the 3-H signal has the width at half-height of 24 Hz, which clearly indicates that it is axial, and therefore, 2-H must be equatorial because its width at half-height is small (10 Hz). The downfield shift of C-1 (78.4) shows that the configuration of the OH group is β (axial), and therefore, 1-H is equatorial. The correlation pattern inferred from COSY, HSQC, and HMBC spectra of compound 9 allows us to state that phenylselenyl group is bonded to C-1. Proton 3-H clearly has configuration β (equatorial) since its width at half-height is 8 Hz. In turn, 2-H has configuration α (equatorial) since long range coupling to 4-H eq is observed in COSY spectrum filling the marks of the H-C-C-C-H W-shape structure ( Figure 2) [30].
the 3-H signal has the width at half-height of 24 Hz, which clearly indicates that it is axial, and therefore, 2-H must be equatorial because its width at half-height is small (10 Hz). The downfield shift of C-1 (78.4) shows that the configuration of the OH group is β (axial), and therefore, 1-H is equatorial. The correlation pattern inferred from COSY, HSQC, and HMBC spectra of compound 9 allows us to state that phenylselenyl group is bonded to C-1. Proton 3-H clearly has configuration β (equatorial) since its width at half-height is 8 Hz. In turn, 2-H has configuration α (equatorial) since long range coupling to 4-Heq is observed in COSY spectrum filling the marks of the H-C-C-C-H Wshape structure ( Figure 2) [30].    the 3-H signal has the width at half-height of 24 Hz, which clearly indicates that it is axial, and therefore, 2-H must be equatorial because its width at half-height is small (10 Hz). The downfield shift of C-1 (78.4) shows that the configuration of the OH group is β (axial), and therefore, 1-H is equatorial. The correlation pattern inferred from COSY, HSQC, and HMBC spectra of compound 9 allows us to state that phenylselenyl group is bonded to C-1. Proton 3-H clearly has configuration β (equatorial) since its width at half-height is 8 Hz. In turn, 2-H has configuration α (equatorial) since long range coupling to 4-Heq is observed in COSY spectrum filling the marks of the H-C-C-C-H Wshape structure ( Figure 2) [30].  the 3-H signal has the width at half-height of 24 Hz, which clearly indicates that it is axial, and therefore, 2-H must be equatorial because its width at half-height is small (10 Hz). The downfield shift of C-1 (78.4) shows that the configuration of the OH group is β (axial), and therefore, 1-H is equatorial. The correlation pattern inferred from COSY, HSQC, and HMBC spectra of compound 9 allows us to state that phenylselenyl group is bonded to C-1. Proton 3-H clearly has configuration β (equatorial) since its width at half-height is 8 Hz. In turn, 2-H has configuration α (equatorial) since long range coupling to 4-Heq is observed in COSY spectrum filling the marks of the H-C-C-C-H Wshape structure ( Figure 2) [30].  the 3-H signal has the width at half-height of 24 Hz, which clearly indicates that it is axial, and therefore, 2-H must be equatorial because its width at half-height is small (10 Hz). The downfield shift of C-1 (78.4) shows that the configuration of the OH group is β (axial), and therefore, 1-H is equatorial. The correlation pattern inferred from COSY, HSQC, and HMBC spectra of compound 9 allows us to state that phenylselenyl group is bonded to C-1. Proton 3-H clearly has configuration β (equatorial) since its width at half-height is 8 Hz. In turn, 2-H has configuration α (equatorial) since long range coupling to 4-Heq is observed in COSY spectrum filling the marks of the H-C-C-C-H Wshape structure ( Figure 2) [30].   Based on these spectroscopic evidence, stereogenic centers at C-1 and C-2 have the absolute configuration depicted in structure 7 and 9, and a plausible mechanism for their formation starting from 1,2-epoxysteroids 6 and 8 with PhSeZnCl is proposed in Scheme 2. In both cases, the nucleophilic attack proceeds in order to minimize the steric hindrance of the approaching selenium reagent with the substituents, following a pseudo-axial attack according to the Fürst-Plattner rule [31]. Furthermore, the interaction of zinc with the hydroxyl group at C-3 could increase the selenium nucleophilicity and cooperate on driving the observed regioselectivity. Steric factors and the lack of a suitable pseudo-axial approach resulted to be particularly detrimental in the reactivity of PhSeZnCl with the epoxy-steroids and could explain all the observed failures. Scheme 2. The proposed mechanism for ring opening of 6 and 8 according to a pseudo-axial ring opening approach.

Biological Activity
A further purpose of this study was to investigate the biological properties of the prepared selenosteroids. The search for new compounds that exhibit antimicrobial properties is a big challenge and an emerging need in modern medicine, especially in the context of the growing number of infections caused by multidrug-resistant bacteria. Furthermore, the antibacterial activity against the biofilm formation of Pseudomonas aeruginosa, which usually causes opportunistic infections, particularly in immunocompromised patients, can be considered a promising characteristic for the development of a new class of antibiotics [32].
The changes of Pseudomonas aeruginosa, Xen 5 luminescence provide an easy way to assess bacteria cell viability and metabolic function. [33] Accordingly, Figure 3A-E shows that all tested agents affect the viability of employed bacteria strain. However, activity of compound 7 was stronger Based on these spectroscopic evidence, stereogenic centers at C-1 and C-2 have the absolute configuration depicted in structure 7 and 9, and a plausible mechanism for their formation starting from 1,2-epoxysteroids 6 and 8 with PhSeZnCl is proposed in Scheme 2. In both cases, the nucleophilic attack proceeds in order to minimize the steric hindrance of the approaching selenium reagent with the substituents, following a pseudo-axial attack according to the Fürst-Plattner rule [31]. Furthermore, the interaction of zinc with the hydroxyl group at C-3 could increase the selenium nucleophilicity and cooperate on driving the observed regioselectivity. Steric factors and the lack of a suitable pseudo-axial approach resulted to be particularly detrimental in the reactivity of PhSeZnCl with the epoxy-steroids and could explain all the observed failures. Scheme 2. The proposed mechanism for ring opening of 6 and 8 according to a pseudo-axial ring opening approach.

Biological Activity
A further purpose of this study was to investigate the biological properties of the prepared selenosteroids. The search for new compounds that exhibit antimicrobial properties is a big challenge and an emerging need in modern medicine, especially in the context of the growing number of infections caused by multidrug-resistant bacteria. Furthermore, the antibacterial activity against the biofilm formation of Pseudomonas aeruginosa, which usually causes opportunistic infections, particularly in immunocompromised patients, can be considered a promising characteristic for the development of a new class of antibiotics [32].
The changes of Pseudomonas aeruginosa, Xen 5 luminescence provide an easy way to assess bacteria cell viability and metabolic function. [33] Accordingly, Figure 3A-E shows that all tested agents affect the viability of employed bacteria strain. However, activity of compound 7 was stronger Based on these spectroscopic evidence, stereogenic centers at C-1 and C-2 have the absolute configuration depicted in structure 7 and 9, and a plausible mechanism for their formation starting from 1,2-epoxysteroids 6 and 8 with PhSeZnCl is proposed in Scheme 2. In both cases, the nucleophilic attack proceeds in order to minimize the steric hindrance of the approaching selenium reagent with the substituents, following a pseudo-axial attack according to the Fürst-Plattner rule [31]. Furthermore, the interaction of zinc with the hydroxyl group at C-3 could increase the selenium nucleophilicity and cooperate on driving the observed regioselectivity. Steric factors and the lack of a suitable pseudo-axial approach resulted to be particularly detrimental in the reactivity of PhSeZnCl with the epoxy-steroids and could explain all the observed failures. Scheme 2. The proposed mechanism for ring opening of 6 and 8 according to a pseudo-axial ring opening approach.

Biological Activity
A further purpose of this study was to investigate the biological properties of the prepared selenosteroids. The search for new compounds that exhibit antimicrobial properties is a big challenge and an emerging need in modern medicine, especially in the context of the growing number of infections caused by multidrug-resistant bacteria. Furthermore, the antibacterial activity against the biofilm formation of Pseudomonas aeruginosa, which usually causes opportunistic infections, particularly in immunocompromised patients, can be considered a promising characteristic for the development of a new class of antibiotics [32].
The changes of Pseudomonas aeruginosa, Xen 5 luminescence provide an easy way to assess bacteria cell viability and metabolic function. [33] Accordingly, Figure 3A-E shows that all tested agents affect the viability of employed bacteria strain. However, activity of compound 7 was stronger when compared to other tested agents. More than 95% of decrease in P. aeruginosa Xen 5 Based on these spectroscopic evidence, stereogenic centers at C-1 and C-2 have the absolute configuration depicted in structure 7 and 9, and a plausible mechanism for their formation starting from 1,2-epoxysteroids 6 and 8 with PhSeZnCl is proposed in Scheme 2. In both cases, the nucleophilic attack proceeds in order to minimize the steric hindrance of the approaching selenium reagent with the substituents, following a pseudo-axial attack according to the Fürst-Plattner rule [31]. Furthermore, the interaction of zinc with the hydroxyl group at C-3 could increase the selenium nucleophilicity and cooperate on driving the observed regioselectivity. Steric factors and the lack of a suitable pseudo-axial approach resulted to be particularly detrimental in the reactivity of PhSeZnCl with the epoxy-steroids and could explain all the observed failures. Scheme 2. The proposed mechanism for ring opening of 6 and 8 according to a pseudo-axial ring opening approach.

Biological Activity
A further purpose of this study was to investigate the biological properties of the prepared selenosteroids. The search for new compounds that exhibit antimicrobial properties is a big challenge and an emerging need in modern medicine, especially in the context of the growing number of infections caused by multidrug-resistant bacteria. Furthermore, the antibacterial activity against the biofilm formation of Pseudomonas aeruginosa, which usually causes opportunistic infections, particularly in immunocompromised patients, can be considered a promising characteristic for the development of a new class of antibiotics [32].
The changes of Pseudomonas aeruginosa, Xen 5 luminescence provide an easy way to assess bacteria cell viability and metabolic function. [33] Accordingly, Figure 3A-E shows that all tested agents affect the viability of employed bacteria strain. However, activity of compound 7 was stronger when compared to other tested agents. More than 95% of decrease in P. aeruginosa Xen 5 Based on these spectroscopic evidence, stereogenic centers at C-1 and C-2 have the absolute configuration depicted in structure 7 and 9, and a plausible mechanism for their formation starting from 1,2-epoxysteroids 6 and 8 with PhSeZnCl is proposed in Scheme 2. In both cases, the nucleophilic attack proceeds in order to minimize the steric hindrance of the approaching selenium reagent with the substituents, following a pseudo-axial attack according to the Fürst-Plattner rule [31]. Furthermore, the interaction of zinc with the hydroxyl group at C-3 could increase the selenium nucleophilicity and cooperate on driving the observed regioselectivity. Steric factors and the lack of a suitable pseudo-axial approach resulted to be particularly detrimental in the reactivity of PhSeZnCl with the epoxy-steroids and could explain all the observed failures. Based on these spectroscopic evidence, stereogenic centers at C-1 and C-2 have the absolute configuration depicted in structure 7 and 9, and a plausible mechanism for their formation starting from 1,2-epoxysteroids 6 and 8 with PhSeZnCl is proposed in Scheme 2. In both cases, the nucleophilic attack proceeds in order to minimize the steric hindrance of the approaching selenium reagent with the substituents, following a pseudo-axial attack according to the Fürst-Plattner rule [31]. Furthermore, the interaction of zinc with the hydroxyl group at C-3 could increase the selenium nucleophilicity and cooperate on driving the observed regioselectivity. Steric factors and the lack of a suitable pseudo-axial approach resulted to be particularly detrimental in the reactivity of PhSeZnCl with the epoxy-steroids and could explain all the observed failures. Scheme 2. The proposed mechanism for ring opening of 6 and 8 according to a pseudo-axial ring opening approach.

Biological Activity
A further purpose of this study was to investigate the biological properties of the prepared selenosteroids. The search for new compounds that exhibit antimicrobial properties is a big challenge and an emerging need in modern medicine, especially in the context of the growing number of infections caused by multidrug-resistant bacteria. Furthermore, the antibacterial activity against the biofilm formation of Pseudomonas aeruginosa, which usually causes opportunistic infections, particularly in immunocompromised patients, can be considered a promising characteristic for the 6

Biological Activity
A further purpose of this study was to investigate the biological properties of the prepared selenosteroids. The search for new compounds that exhibit antimicrobial properties is a big challenge and an emerging need in modern medicine, especially in the context of the growing number of infections caused by multidrug-resistant bacteria. Furthermore, the antibacterial activity against the biofilm formation of Pseudomonas aeruginosa, which usually causes opportunistic infections, particularly in immunocompromised patients, can be considered a promising characteristic for the development of a new class of antibiotics [32].
The changes of Pseudomonas aeruginosa, Xen 5 luminescence provide an easy way to assess bacteria cell viability and metabolic function. [33] Accordingly, Figure 3A-E shows that all tested agents affect the viability of employed bacteria strain. However, activity of compound 7 was stronger when compared to other tested agents. More than 95% of decrease in P. aeruginosa Xen 5 chemiluminescence, was observed after 10 min of incubation when highest dose of 7 was tested. This inhibitory effect is comparable to activity of standard antibiotic agents used in concentrations corresponding to a 100-fold increase of MIC value ( Figure 3E). The obtained results suggest that the position of phenylselenyl group might affect and modulate the antimicrobial activity. Additionally, the activity of synthesized compounds, was compared to that of colistin, currently used in the treatment of P. aeruginosa infections. Interestingly, it was observed that the use of colistin at a dose corresponding to 1xMIC value does not affect the metabolic function of P. aeruginosa infections caused by MDR strains. In turn, application of 5-fold MIC concentration disturbs the cell function at 60%. the activity of synthesized compounds, was compared to that of colistin, currently used in the treatment of P. aeruginosa infections. Interestingly, it was observed that the use of colistin at a dose corresponding to 1xMIC value does not affect the metabolic function of P. aeruginosa infections caused by MDR strains. In turn, application of 5-fold MIC concentration disturbs the cell function at 60%. In a different set of experiments, luminometric measurements was applied to determine the ability of the tested phenylselenium-substituted steroids on preventing bacterial biofilm formation. Figure 4A-C illustrates that the tested agents are able to inhibit biofilm formation and effectively kill bacteria embedded into the biofilm matrix in time and dose-dependent manners. However, after 24 h, in the case of compound 7, high doses (>50 μg/mL) were required to obtain ~50% inhibition of biofilm formation. In the case of mature biofilm, formed after 48 and 72 h treatment with the tested agents (> 20 μg/mL), decreases biofilm viability by ~50%, for compound 2 and 4 respectively, and ~ 90% for compound 7. In the case of colistin, their use in 1 and 5-fold MIC was insufficient to restrict biofilm metabolic activity. Therefore, the observed proprieties of the tested agents might help in developing of effective strategies against Pseudomonas aeruginosa biofilm formation, which is directly associated with hospital infections via the colonization of medical devices, as well as a major cause of the recurrence and chronic infections, such as pneumonia in cystic fibrosis patients. Due to amphipathic nature of tested phenylselenium-substituted steroids and their similarity to ceragenins, which possess a broad spectrum of antimicrobial activity, further studies are needed to determine a full antimicrobial spectrum of the tested agents and to establish their mode of action [34].  In a different set of experiments, luminometric measurements was applied to determine the ability of the tested phenylselenium-substituted steroids on preventing bacterial biofilm formation. Figure 4A-C illustrates that the tested agents are able to inhibit biofilm formation and effectively kill bacteria embedded into the biofilm matrix in time and dose-dependent manners. However, after 24 h, in the case of compound 7, high doses (>50 µg/mL) were required to obtain~50% inhibition of biofilm formation. In the case of mature biofilm, formed after 48 and 72 h treatment with the tested agents (> 20 µg/mL), decreases biofilm viability by~50%, for compound 2 and 4 respectively, and~90% for compound 7. In the case of colistin, their use in 1 and 5-fold MIC was insufficient to restrict biofilm metabolic activity. Therefore, the observed proprieties of the tested agents might help in developing of effective strategies against Pseudomonas aeruginosa biofilm formation, which is directly associated with hospital infections via the colonization of medical devices, as well as a major cause of the recurrence and chronic infections, such as pneumonia in cystic fibrosis patients. Due to amphipathic nature of tested phenylselenium-substituted steroids and their similarity to ceragenins, which possess a broad spectrum of antimicrobial activity, further studies are needed to determine a full antimicrobial spectrum of the tested agents and to establish their mode of action [34].  The analysis of 77 Se-NMR chemical shift of 2,4,7 and 9 (reported in Figure 5) afforded interesting consideration. In compounds 7 and 9 the presence of hydroxyl groups in a suitable position to establish a non-bonding interaction with the selenium atom produce an evident upfield of the chemical shift (−60 ppm for 9; −116 ppm for 7 respect 2 and 4). This correspond to a higher electron density on the selenium atom of 7 respect to the other derivatives and, consequently, a lower electrophilicity to which correspond a reduced prooxidant activity and, reasonably, a lower general toxicity.  The analysis of 77 Se-NMR chemical shift of 2,4,7 and 9 (reported in Figure 5) afforded interesting consideration. In compounds 7 and 9 the presence of hydroxyl groups in a suitable position to establish a non-bonding interaction with the selenium atom produce an evident upfield of the chemical shift (−60 ppm for 9; −116 ppm for 7 respect 2 and 4). This correspond to a higher electron density on the selenium atom of 7 respect to the other derivatives and, consequently, a lower electrophilicity to which correspond a reduced prooxidant activity and, reasonably, a lower general toxicity. Considering that Rodrigues et al. reported that derivatives similar to 2 are characterized by an important prooxidant activity we explored the redox behavior of 2 as well as its GPx-like activity using the 77 Se-NMR and NMR-DTT coupled test [16,17]. Initially, we investigated by 77 Se-NMR spectroscopy, the actual intermediates involved in GPx-like cycle. With the addition of hydrogen peroxide (5 eq), we observed the formation of a couple of diastereomeric selenoxides (δ 77 Se-NMR in CD3OD: 901 and 866 ppm), that can be readily reduced to selenide by the addition of a stoichiometric amount (5 eq) of reduced dithiotreitol (DTTred). The experiment was performed directly into the NMR tube (Scheme 3). The kinetics of the peroxide reduction catalyzed by 2 was measured studying via 1 H-NMR the oxidation of DTT. This process resulted particularly slow producing, after 19 h, only 22% of oxidized DTT. This value is particularly low respect those reported in literature for other GPx mimetics and we believe that it could be due to the steric hindrance around the selenium atom of selenoxide, that prevented the fast attack of the thiol and consequently the reduction of the selenoxide.  Considering that Rodrigues et al. reported that derivatives similar to 2 are characterized by an important prooxidant activity we explored the redox behavior of 2 as well as its GPx-like activity using the 77 Se-NMR and NMR-DTT coupled test [16,17]. Initially, we investigated by 77 Se-NMR spectroscopy, the actual intermediates involved in GPx-like cycle. With the addition of hydrogen peroxide (5 eq), we observed the formation of a couple of diastereomeric selenoxides (δ 77 Se-NMR in CD 3 OD: 901 and 866 ppm), that can be readily reduced to selenide by the addition of a stoichiometric amount (5 eq) of reduced dithiotreitol (DTTred). The experiment was performed directly into the NMR tube (Scheme 3). The kinetics of the peroxide reduction catalyzed by 2 was measured studying via 1 H-NMR the oxidation of DTT. This process resulted particularly slow producing, after 19 h, only 22% of oxidized DTT. This value is particularly low respect those reported in literature for other GPx mimetics and we believe that it could be due to the steric hindrance around the selenium atom of selenoxide, that prevented the fast attack of the thiol and consequently the reduction of the selenoxide.

General Methods
Reagent-grade chemicals were purchased and used as received. Methylene chloride was freshly distilled. Flash column chromatography and flash chromatography were performed with silica gel, pore size 40A (70-230 mesh), unless otherwise stated. All reactions were monitored by TLC on silica gel plates 60 F254. 1 H (400 MHz) and 13 C NMR (100 MHz) spectra for all compounds were recorded at ambient temperature and were referenced to TMS (0.0 ppm) and CDCl3 (77.0 ppm), respectively, unless otherwise noted. NMR resonance multiplicities were reported using the following abbreviations: b = broad, s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet; coupling constants J were reported in Hz. IR spectra were obtained in a CHCl3 solution with an FT-IR spectrometer, and data are reported in cm −1 . Melting points were determined by a Kofler bench (Boetius type) apparatus and are uncorrected. (Spectra of the synthetized compounds are collected in the Supplementary materials)

General Procedure
To a solution of the PhSeCl (1.0 equiv.) in anhydrous THF (1 mL/mmol) under argon atmosphere and at reflux, the activated zinc (1.0 equiv) was added. The formation of PhSeZnCl was indicated by the formation of colourless solution. Then, a solution of the starting material (1.0 equiv) in THF under argon atmosphere was added. The reaction was stirred at reflux for 3-8 h. Then THF was removed under vacuum. The yellow oil obtained was dissolved in CH2Cl2 and washed 3 times with H2O. The organic layers were dried with Na2SO4, filtered and the solvent removed under vacuum. The products were purified by flash chromatography.

General Methods
Reagent-grade chemicals were purchased and used as received. Methylene chloride was freshly distilled. Flash column chromatography and flash chromatography were performed with silica gel, pore size 40A (70-230 mesh), unless otherwise stated. All reactions were monitored by TLC on silica gel plates 60 F 254 . 1 H (400 MHz) and 13 C NMR (100 MHz) spectra for all compounds were recorded at ambient temperature and were referenced to TMS (0.0 ppm) and CDCl 3 (77.0 ppm), respectively, unless otherwise noted. NMR resonance multiplicities were reported using the following abbreviations: b = broad, s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet; coupling constants J were reported in Hz. IR spectra were obtained in a CHCl 3 solution with an FT-IR spectrometer, and data are reported in cm −1 . Melting points were determined by a Kofler bench (Boetius type) apparatus and are uncorrected. (Spectra of the synthetized compounds are collected in the Supplementary Materials)

General Procedure
To a solution of the PhSeCl (1.0 equiv.) in anhydrous THF (1 mL/mmol) under argon atmosphere and at reflux, the activated zinc (1.0 equiv) was added. The formation of PhSeZnCl was indicated by the formation of colourless solution. Then, a solution of the starting material (1.0 equiv) in THF under argon atmosphere was added. The reaction was stirred at reflux for 3-8 h. Then THF was removed under vacuum. The yellow oil obtained was dissolved in CH 2 Cl 2 and washed 3 times with H 2 O. The organic layers were dried with Na 2 SO 4 , filtered and the solvent removed under vacuum. The products were purified by flash chromatography.

Antibacterial Testing
To assess the biological activities of synthesized compounds, we have chosen to determine their antibacterial activity against Gram-negative, multidrug resistant (MDR) Pseudomonas aeruginosa Xen 5 strain. Briefly, a bacterial culture was grown to mid-log phase at 37 • C, re-suspended in LB, and brought to 10 8 CFU/mL. Then 100 µL of bacteria suspensions were added to tested agents at a concentration range 1-100 µg/mL. Colistin was use as a control, since it represents "last treatment option" in some infections caused by MDR Pseudomonas aeruginosa. Upon bacteria addition the chemiluminescence intensity were registered using Labsystems Varioscan Flash (Thermo Scientific Waltham, MA, USA) over a period of 1h.

Anti-Biofilm Activity
The biomass of biofilm, formed by Pseudomonas aeruginosa Xen 5 in the presence of tested molecules, was evaluated using a luminescence technique as described previously [39]. Chemiluminescence intensity of Pseudomonas aeruginosa Xen 5 biomass was measured after 24, 48, and 72 h of growth. In this setting, time-dependent biofilm mass and viability was determined

GPx-Like Activity by NMR
In a 5mm NMR tube containing 0.15 mmol of DTT red and 0.015 of catalyst (2) dissolved in 0.55 mL of CD 3 OD, 0.15 mmol of H 2 O 2 (30%) was added and 1 H-NMR experiments at 200 MHz were recorded at regular interval for a period of 24 h. Ratios between DTT red /DTT ox were determined by comparison of integrals at 2.88 ppm and 2.64 ppm, respectively.

Conclusions
In conclusion we demonstrated that in situ formed PhSeZnCl can be used for the functionalization of steroids with selenium moieties. The obtained novel compounds were tested for their potential antibacterial properties against Pseudomonas aeruginosa Xen 5 strain, evidencing a promising bactericidal activity associated to a biofilm formation prevention.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.