Phenylselanyl Group Incorporation for “Glutathione Peroxidase-Like” Activity Modulation

The ability of organoselenium molecules to mimic the activity of the antioxidant selenoenzyme glutathione peroxidase (GPx) allows for their use as antioxidant or prooxidant modulators in several diseases associated with the disruption of the cell redox homeostasis. Current drug design in the field is partially based on specific modifications of the known Se-therapeutics aimed at achieving more selective bioactivity towards particular drug targets, accompanied by low toxicity as the therapeutic window for organoselenium compounds tends to be very narrow. Herein, we present a new group of Se-based antioxidants, structurally derived from the well-known group of GPx mimics—benzisoselenazol-3(2H)-ones. A series of N-substituted unsymmetrical phenylselenides with an o-amido function has been obtained by a newly developed procedure: a copper-catalyzed nucleophilic substitution by a Se-reagent formed in situ from diphenyl diselenide and sodium borohydride. All derivatives were tested as antioxidants and anticancer agents towards breast (MCF-7) and leukemia (HL-60) cancer cell lines. The highest H2O2-scavenging potential was observed for N-(3-methylbutyl)-2-(phenylselanyl)benzamide. The best antiproliferative activity was found for (−)-N-(1S,2R,4R)-menthyl-2-(phenylselanyl)benzamide (HL-60) and ((−)-N-(1S,2R,3S,6R)-(2-caranyl))benzamide (MCF-7). The structure–activity correlations, including the differences in reactivity of the obtained phenyl selenides and corresponding benzisoselenazol-3(2H)-ones, were performed.


Introduction
Drug design is a multi-step process, focused on the obtainment of the most specific ligand-receptor interaction correlated to a suitable structural core that is able to equip the molecule with a potential biological activity. The first discovered lead compound is subsequently variously functionalized in order to increase (and maximize) the desired therapeutic activity over the toxicity. The pharmacophore modeling often includes installation of aromatic or heteroaromatic rings, which are easy to introduce, can be further manipulated and are often responsible of the activity [1,2].
In the field of organoselenium chemistry, the design of Se-based therapeutics is often connected with the ability of selenium pharmacophores to mimic the activity of glutathione peroxidase (GPx). Over the years, the role of organoselenium compounds as redox-modulators was well-established with numerous examples of biologically active molecules [3][4][5][6], including the antioxidant agent with numerous examples of biologically active molecules [3][4][5][6], including the antioxidant agent Nphenylbenzisoselenazol-3(2H)-one (named as Ebselen) 1, currently in phase II clinical trial for noiseinduced hearing loss [7]. Similarly, to ebselen 1, a significant number of proven bioactive Semolecules possess aromatic or heteroaromatic rings as the core of the molecule [3]. Examples are presented in Scheme 1 and also include other Se-therapeutics currently in clinical trial: ethaselen 2, Trx reductase inhibitor, antitumor agent [8] and 4,4-dimethyl-benziso-2H-selenazine 3, antiinflammatory therapeutic tested in chronic plaque psoriasis [9]. Many research groups continue the study of new strategies and structural modifications to obtain new Se-antioxidants that have high and selective activity. In our previous work, we explored the possibility to improve the GPx-like activity of ebselen with the introduction of specific functionalities that would enable new highly efficient biocatalyst [10][11][12]. Various N-aromatic and Naliphatic derivatives 4 were obtained and easily transformed into the corresponding diselenides 5 (path a) [13][14][15] and seleninic acids, as well as to their potassium salts 6 (path b) [16]. Determination of the antioxidant and antiproliferative potential of all obtained molecules revealed a particular structure-activity relationship. Besides the observed influence of the N-substituent on their biological potential, it was also recently highlighted by Santi and co-workers [17], that the form of the Se-moiety is crucial for the specific catalytic activity of the designed GPx-mimics. To further differentiate the structures and to broaden the scope of the tested molecules, we introduced a phenylselanyl group as a new benzisoselenazolone core modification (7b-23b, Scheme 2). This modification allowed us to obtain a large group of GPx mimetics 7b-23b and to determine whether the introduction of an additional aromatic ring and the exchange of Se-N for Se-Car bond is justified in order to obtain higher therapeutic potential of the N-substituted ebselen-like antioxidants.

Results and Discussion
The first step of the research involved the synthesis of N-substituted o-iodobenzamides 7a-23a. The compounds were obtained through the reaction of the corresponding amines with o-iodobenzoic acid chloride 8. Benzamides 7a-23a were further transformed to the final N-aliphatic 7b-12b, N-Scheme 1. Bioactive organoselenium compounds 1-3 that possess aromatic rings in their structure.
Many research groups continue the study of new strategies and structural modifications to obtain new Se-antioxidants that have high and selective activity. In our previous work, we explored the possibility to improve the GPx-like activity of ebselen with the introduction of specific functionalities that would enable new highly efficient biocatalyst [10][11][12]. Various N-aromatic and N-aliphatic derivatives 4 were obtained and easily transformed into the corresponding diselenides 5 (path a) [13][14][15] and seleninic acids, as well as to their potassium salts 6 (path b) [16]. Determination of the antioxidant and antiproliferative potential of all obtained molecules revealed a particular structure-activity relationship. Besides the observed influence of the N-substituent on their biological potential, it was also recently highlighted by Santi and co-workers [17], that the form of the Se-moiety is crucial for the specific catalytic activity of the designed GPx-mimics. To further differentiate the structures and to broaden the scope of the tested molecules, we introduced a phenylselanyl group as a new benzisoselenazolone core modification (7b-23b, Scheme 2). with numerous examples of biologically active molecules [3][4][5][6], including the antioxidant agent Nphenylbenzisoselenazol-3(2H)-one (named as Ebselen) 1, currently in phase II clinical trial for noiseinduced hearing loss [7]. Similarly, to ebselen 1, a significant number of proven bioactive Semolecules possess aromatic or heteroaromatic rings as the core of the molecule [3]. Examples are presented in Scheme 1 and also include other Se-therapeutics currently in clinical trial: ethaselen 2, Trx reductase inhibitor, antitumor agent [8] and 4,4-dimethyl-benziso-2H-selenazine 3, antiinflammatory therapeutic tested in chronic plaque psoriasis [9]. Many research groups continue the study of new strategies and structural modifications to obtain new Se-antioxidants that have high and selective activity. In our previous work, we explored the possibility to improve the GPx-like activity of ebselen with the introduction of specific functionalities that would enable new highly efficient biocatalyst [10][11][12]. Various N-aromatic and Naliphatic derivatives 4 were obtained and easily transformed into the corresponding diselenides 5 (path a) [13][14][15] and seleninic acids, as well as to their potassium salts 6 (path b) [16]. Determination of the antioxidant and antiproliferative potential of all obtained molecules revealed a particular structure-activity relationship. Besides the observed influence of the N-substituent on their biological potential, it was also recently highlighted by Santi and co-workers [17], that the form of the Se-moiety is crucial for the specific catalytic activity of the designed GPx-mimics. To further differentiate the structures and to broaden the scope of the tested molecules, we introduced a phenylselanyl group as a new benzisoselenazolone core modification (7b-23b, Scheme 2). This modification allowed us to obtain a large group of GPx mimetics 7b-23b and to determine whether the introduction of an additional aromatic ring and the exchange of Se-N for Se-Car bond is justified in order to obtain higher therapeutic potential of the N-substituted ebselen-like antioxidants.

Results and Discussion
The first step of the research involved the synthesis of N-substituted o-iodobenzamides 7a-23a. The compounds were obtained through the reaction of the corresponding amines with o-iodobenzoic acid chloride 8. Benzamides 7a-23a were further transformed to the final N-aliphatic 7b-12b, N- This modification allowed us to obtain a large group of GPx mimetics 7b-23b and to determine whether the introduction of an additional aromatic ring and the exchange of Se-N for Se-C ar bond is justified in order to obtain higher therapeutic potential of the N-substituted ebselen-like antioxidants.

Results and Discussion
The first step of the research involved the synthesis of N-substituted o-iodobenzamides 7a-23a. The compounds were obtained through the reaction of the corresponding amines with o-iodobenzoic acid chloride 8. Benzamides 7a-23a were further transformed to the final N-aliphatic 7b-12b, thiol cofactor. The conversion of the dithiol DTT red to the disulphide DTT ox was observed in 1 HNMR spectra in the specific time intervals. The results for the most active derivatives are presented in Scheme 5. The results obtained for all compounds are reported in Supporting Information. The highest antioxidant potential was observed for N-butyl 9b, N-3-methylbutyl 10b and Npinanyl phenyl selenide 22b. The results of the three selected Se-catalysts were compared to corresponding benzisoselenazol-3(2H)-ones 27-29. It could be noticed that the bulkiness of the substituent enhances the H2O2 scavenging activity of benzisoselenazolones (reactivity: 29 > 28 > 27) but decreased it for the corresponding phenylselenides (reactivity: 10b > 22b > 9b). For compounds 27-29, the hindrance of the N-substituent facilitated the cleavage of the Se-N bond that accelerated the Se-moiety oxidation by hydrogen peroxide. On the contrary, the reaction of -SePh group with H2O2 proceeded more efficiently when the alkyl chain did not hinder the selenium atom.
To investigate the mechanism of the antioxidant activity, we have performed an additional 77 Se NMR experiment of the H2O2-oxidation product of the most reactive N-butyl phenylselenide 9b (the sample was stored for 12 h before the NMR recording). A signal at 853 ppm indicated the formation of corresponding selenooxide. Based on these observations, supported by previous literature reports [20,21], we assume that the possible GPx-like catalytic cycle of the tested phenyl selenides involves the formation of the selenoxide 30, which is further hydrated to the corresponding hydrated oxide 31. The final H2O2 reduction and thiol oxidation proceeds through the reversible formation of the peroxy-hydrated oxide 32 (Scheme 6). The highest antioxidant potential was observed for N-butyl 9b, N-3-methylbutyl 10b and N-pinanyl phenyl selenide 22b. The results of the three selected Se-catalysts were compared to corresponding benzisoselenazol-3(2H)-ones 27-29. It could be noticed that the bulkiness of the substituent enhances the H 2 O 2 scavenging activity of benzisoselenazolones (reactivity: 29 > 28 > 27) but decreased it for the corresponding phenylselenides (reactivity: 10b > 22b > 9b). For compounds 27-29, the hindrance of the N-substituent facilitated the cleavage of the Se-N bond that accelerated the Se-moiety oxidation by hydrogen peroxide. On the contrary, the reaction of -SePh group with H 2 O 2 proceeded more efficiently when the alkyl chain did not hinder the selenium atom.
To investigate the mechanism of the antioxidant activity, we have performed an additional 77 Se NMR experiment of the H 2 O 2 -oxidation product of the most reactive N-butyl phenylselenide 9b (the sample was stored for 12 h before the NMR recording). A signal at 853 ppm indicated the formation of corresponding selenooxide. Based on these observations, supported by previous literature reports [20,21], we assume that the possible GPx-like catalytic cycle of the tested phenyl selenides involves the formation of the selenoxide 30, which is further hydrated to the corresponding hydrated oxide 31. The final H 2 O 2 reduction and thiol oxidation proceeds through the reversible formation of the peroxy-hydrated oxide 32 (Scheme 6).

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Molecules 2020, 25, x FOR PEER REVIEW 5 of 13 Scheme 6. Plausible mechanism for the H2O2 reduction by the Se-catalysts in the presence of thiols.
Next, all phenylselenides 7b-23b were evaluated as antiproliferative agents by the MTT (3-(4,5dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay against breast (MCF-7) and leukemia (HL-60) cell lines. The best results were obtained for N-terpene derivatives 18b, 19b and compared with the data for the corresponding benzisoselenazol-3(2H)-ones 33 and 34 (Table 1). For the rest of the tested samples 7b-17b, 20b-23b, the IC50 values were above 50 µM. Additionally, it was observed that the antiproliferative potential of analogs increased when the phenylselanyl moiety was introduced into the structure, showing that an additional aromatic ring can be beneficial for the compound's cytotoxicity.
We have previously noticed that the internal 2-methylbutyl carbon chain is a repetitive element in the structure of the active benzisoselenazol-3(2H)-ones 28, 33 and 35, which indicates its potential role as a pharmacophore. Additional carbon chains or functional groups attached to the 2methylbutyl substituent influenced the inhibitory potential. The antiproliferative activity was the highest for compounds with the carbon chain expanded to the cyclic menthyl functionality, benzisoselenazol-3(2H)-one 30 and phenylselenide 18b with IC50 values 11.9 ± 0.2 µM (MCF-7) and 10.7 ± 0.6 µM (HL-60), respectively (Scheme 7). Next, all phenylselenides 7b-23b were evaluated as antiproliferative agents by the MTT (3-(4,5dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay against breast (MCF-7) and leukemia (HL-60) cell lines. The best results were obtained for N-terpene derivatives 18b, 19b and compared with the data for the corresponding benzisoselenazol-3(2H)-ones 33 and 34 (Table 1). For the rest of the tested samples 7b-17b, 20b-23b, the IC50 values were above 50 µM. Additionally, it was observed that the antiproliferative potential of analogs increased when the phenylselanyl moiety was introduced into the structure, showing that an additional aromatic ring can be beneficial for the compound's cytotoxicity.
We have previously noticed that the internal 2-methylbutyl carbon chain is a repetitive element in the structure of the active benzisoselenazol-3(2H)-ones 28, 33 and 35, which indicates its potential role as a pharmacophore. Additional carbon chains or functional groups attached to the 2methylbutyl substituent influenced the inhibitory potential. The antiproliferative activity was the highest for compounds with the carbon chain expanded to the cyclic menthyl functionality, benzisoselenazol-3(2H)-one 30 and phenylselenide 18b with IC50 values 11.9 ± 0.2 µM (MCF-7) and 10.7 ± 0.6 µM (HL-60), respectively (Scheme 7). Next, all phenylselenides 7b-23b were evaluated as antiproliferative agents by the MTT (3-(4,5dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay against breast (MCF-7) and leukemia (HL-60) cell lines. The best results were obtained for N-terpene derivatives 18b, 19b and compared with the data for the corresponding benzisoselenazol-3(2H)-ones 33 and 34 (Table 1). For the rest of the tested samples 7b-17b, 20b-23b, the IC50 values were above 50 µM. Additionally, it was observed that the antiproliferative potential of analogs increased when the phenylselanyl moiety was introduced into the structure, showing that an additional aromatic ring can be beneficial for the compound's cytotoxicity.
We have previously noticed that the internal 2-methylbutyl carbon chain is a repetitive element in the structure of the active benzisoselenazol-3(2H)-ones 28, 33 and 35, which indicates its potential role as a pharmacophore. Additional carbon chains or functional groups attached to the 2methylbutyl substituent influenced the inhibitory potential. The antiproliferative activity was the highest for compounds with the carbon chain expanded to the cyclic menthyl functionality, benzisoselenazol-3(2H)-one 30 and phenylselenide 18b with IC50 values 11.9 ± 0.2 µM (MCF-7) and 10.7 ± 0.6 µM (HL-60), respectively (Scheme 7). Next, all phenylselenides 7b-23b were evaluated as antiproliferative agents by the MTT (3-(4,5dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay against breast (MCF-7) and leukemia (HL-60) cell lines. The best results were obtained for N-terpene derivatives 18b, 19b and compared with the data for the corresponding benzisoselenazol-3(2H)-ones 33 and 34 (Table 1). For the rest of the tested samples 7b-17b, 20b-23b, the IC50 values were above 50 µM. Additionally, it was observed that the antiproliferative potential of analogs increased when the phenylselanyl moiety was introduced into the structure, showing that an additional aromatic ring can be beneficial for the compound's cytotoxicity.
We have previously noticed that the internal 2-methylbutyl carbon chain is a repetitive element in the structure of the active benzisoselenazol-3(2H)-ones 28, 33 and 35, which indicates its potential role as a pharmacophore. Additional carbon chains or functional groups attached to the 2methylbutyl substituent influenced the inhibitory potential. The antiproliferative activity was the highest for compounds with the carbon chain expanded to the cyclic menthyl functionality, benzisoselenazol-3(2H)-one 30 and phenylselenide 18b with IC50 values 11.9 ± 0.2 µM (MCF-7) and 10.7 ± 0.6 µM (HL-60), respectively (Scheme 7). Additionally, it was observed that the antiproliferative potential of analogs increased when the phenylselanyl moiety was introduced into the structure, showing that an additional aromatic ring can be beneficial for the compound's cytotoxicity.
We have previously noticed that the internal 2-methylbutyl carbon chain is a repetitive element in the structure of the active benzisoselenazol-3(2H)-ones 28, 33 and 35, which indicates its potential role as a pharmacophore. Additional carbon chains or functional groups attached to the 2-methylbutyl substituent influenced the inhibitory potential. The antiproliferative activity was the highest for compounds with the carbon chain expanded to the cyclic menthyl functionality, benzisoselenazol-3(2H)-one 30 and phenylselenide 18b with IC 50 values 11.9 ± 0.2 µM (MCF-7) and 10.7 ± 0.6 µM (HL-60), respectively (Scheme 7).

Synthesis of N-substituted o-iodobenzamides 7a-23a
2% NaOH (4.4 mL) was added to a solution of an amine (1.0 mmol) in DCM (2 mL). The mixture was cooled to 0 • C and o-iodobenzoic acid chloride (1.1 mmol) dissolved in DCM (3 mL) was added dropwise. The reaction mixture was stirred at room temperature for 20 h and the product was extracted with DCM. Combined organic layers were washed with saturated NaHCO 3 and dried over magnesium sulfate. The solvent was removed under reduced pressure and the product was obtained as white solid.  2951, 2916, 2867, 1636, 1584, 1540, 1462, 1430, 1385, 1367, 1341, 1325, 1307, 1261, 1161, 1147, 1116  To a solution of a diphenyl diselenide (0.5 mmol) in dry toluene (5 mL), sodium borohydride (1.5 mmol) was added and stirred at room temperature. Next, DMSO was added dropwise until the solution discolored. Then, respectively, CuI (0.1 mmol), 1,10-phenanthroline (0.2 mmol) and an amide (1.0 mmol) were added. The mixture was stirred under reflux for 18 h. The solution was cooled to room temperature and brine (5 mL) was added. The product was extracted with chloroform (2 × 10 mL), and the combined organic layers were washed with water (2 × 10 mL), brine (2 × 10 mL) and dried over magnesium sulphate. The solvent was removed under reduced pressure and the obtained crude product was isolated by column chromatography (silica gel, DCM