Water-dependent synthesis of biologically active diaryl diselenides

A new one-step method for the synthesis of diaryl diselenides has been developed. The reaction of o - iodobenzamides with dilithium diselenide can be controlled by the presence of water providing a simple and efficient protocol to obtain benzisoselenazolones or diaryl diselenides. A series of N -aryl ebselen derivatives and the corresponding diselenides was obtained. All synthesized compounds were tested in vitro as antioxidants and cytotoxic agents. N -(2,3,4-trimethoxyphenyl)benzisoselenazol-3(2 H )-one was the best in vitro antioxidant and the corresponding diselenide the most potent cytotoxic agent against prostate cancer cell line DU145, being inactive towards healthy prostate cell line PNT1A.


Scheme 2. Approaches to diselenides with amido function 8.
The aim of this study was the synthesis of diaryl diselenides with an amido function substituted on the nitrogen atom by a phenyl ring carrying electron withdrawing or electron donating groups and comparison of their antioxidant activity to the GPx-capacity of the corresponding benzisoselenazolones and ebselen.
Compounds 1, 9-14 were further transformed into the corresponding diselenides 2, 15-20 using sodium borohydride as the reducing agent followed by air oxidation (method C), however, low yields required further studies.During our investigations on the synthesis of ebselen derivatives, based on the reaction of Nsubstituted o-iodobenzamide with dilithium diselenide, we observed that water influences the reaction course leading to the formation of diselenides as by-products.Consequently, a new one-step synthesis of compounds 2, 15-20 was developed (method D).According to our previously proposed mechanism for the formation of ebselen derivatives, in the first reaction step corresponding diselenides are formed, which are then oxidized to benzisoselenazolones. 42 Apparently, the addition of a small amount of water (5%) decreases the solubility of diselenides what prevents further oxidation reactions.Increasing the amount of added water completely inhibited the product formation and the unreacted substrate was isolated after 24 h.Although the newly presented procedure is based on the known reaction of o-iodobenzamides with LiSeSeLi (method i) significant improvements have been achieved.Lithium diselenide is obtained by a modified protocol from selenium and lithium hydroxide in the presence of hydrazine hydrate, and no catalyst is needed for the reaction to proceed. 42Formation of monoselenides in the reaction mixture was not observed. 37Addition of water (5%) to the reaction medium enables to obtain diaryl diselenides as the only products directly from o-iodobenzamides (Scheme 4).

Scheme 4. Water-controlled synthesis of diaryl diselenides.
Results for the synthesis of diaryl diselenides 2, 15-20 by both methods C and D are collected in Table 1.
Reducing the procedure to one step increased the overall yields of the reactions.The newly developed waterdependent methodology was significantly more efficient than the synthesis of benzisoselenazolone and its further reduction and air oxidation.
All of the obtained benzisoselenazolones and diselenides were evaluated as in vitro antioxidants using the NMR assay proposed by Iwaoka. 45Time of conversion of dithiol (DTT red ) to a disulphide (DTT ox ) in the presence of H2O2 and 10% of selenocatalyst was measured in selected time intervals (Table 2).The highest activity was obtained for benzisoselenazolone 14 with an additional methoxy moiety.In this case no substrate was observed after 5 min of the reaction time.All derivatives were also tested as cytotoxic agents towards prostate cancer cell line DU145.Results are collected in Table 3.The highest cytotoxic activity against cancer cells was observed for diselenides bearing additional methoxy substituents in the N-phenyl ring 15 and 20.Compounds exhibiting cytotoxic potential were additionally tested on normal cell line PNT1A.The data showed that both compounds demonstrated low toxicity towards PNT1A cells.They were also more active than commercially used cisplatin.Conversely, compounds 9 and 2 exhibited the same cytotoxic activity against noncancerous and cancer cells.

Conclusions
Herein, we have established that by adding 5 % of water we can initiate the formation of a Se-Se instead of Se-N bond.Simple modification of the previously presented methodology to synthesize benzisoselenazolones led to the development of an efficient protocol to obtain diaryl diselenides with amido function.A series of diselenides was synthesized by two methods: from the reduction and further air oxidation of previously obtained benzisoselenazolones and by the newly developed methodology which was more efficient.An additional advantage of the new methodology is the lack of monoselenides as the side products.All compounds were tested as in vitro antioxidants and cytotoxic agents.The highest in vitro antioxidant activity was obtained for benzisoselenazolone 14 for which no substrate was observed after 5 min.The cytotoxic activity assay performed on prostate cancer cell line DU145 showed that the presence of the methoxy substituent, also attached to the N-phenyl ring of compound 14, influences the biological activity of derivatives 15 and 20 with the highest cytotoxic potential.It is important to note that both compounds 15 and 20 showed specific activity against cancer cells higher than cisplatin but not against normal epithelial cell line PNT1A.Thus, it can be concluded that both compounds are worth of further study as potential drugs.

Experimental Section
General. 1 H NMR spectra were obtained at 400 or 700 MHz and chemical shifts were recorded relative to SiMe4 ( 0.00) or solvent resonance (CDCl3  7.26, CD3OD  3.31).Multiplicities were given as: s (singlet), d (doublet), dd (double doublet), ddd (double double doublet), t (triplet), td (triple doublet), dt (double triplet) and m (multiplet).The number of protons (n) for a given resonance was indicated by nH.Coupling constants were reported as a J value in Hz. 13 C NMR spectra were acquired at 100.6 MHz and chemical shifts were recorded relative to solvent resonance (CDCl3 77.25).NMR spectra were carried out using ACD/NMR Processor Academic Edition.Commercially available solvents DMF, DCM and MeOH (Aldrich) and chemicals were used without further purification.Column chromatography was performed using Merck 40-63D 60Å silica gel.

Synthesis of compounds 1, 9-14
Method A. 44 Preparation of 2-(chloroseleno)benzoyl chloride.To a suspension of selenium (12.7 mmol) in water (7 mL) sodium borohydride (25.3 mmol) was added under argon atmosphere.Mixture was stirred for 0.5h at 0 o C, selenium (12.7 mmol) was added, and stirring was continued at the same temperature for 1h.Mixture was warmed to room temperature and stirred for 18 h.After adding 40% NaOH (5 mL) the reaction was cooled to 5 o C and the diazonium salt of anthranilic acid was added dropwise.(The diazonium salt was prepared z in advance -to a solution of anthranilic acid (26.0 mmol) and concentrated hydrochloric acid (5 mL) in water (15 mL) cooled to 5 o C a cooled solution of sodium nitrite (27.5 mmol) in water (15 mL) was added dropwise and the reaction was stirred for 15 min at 5 o C).The mixture was stirred for 3 h at 60 o C and for 18 h at room temperature.The formed precipitate was filtered off and the solution was acidified to pH = 1 by 36% HCl.The formed precipitate was filtrated.The crude product was purified by washing with boiling water and dried in air.Acid (10.0 mmol) was further converted to 2-(chloroseleno)benzoyl chloride by heating with thionyl chloride (20 mL) at 85 o C for 3 h.Thionyl chloride was distilled off, and the crude product was used without further purification.Preparation of benzisoselenazlones.To a solution of amine (2.0 mmol) and triethylamine (4.0 mmol) in dichloromethane 2-(chloroseleno)benzoyl chloride (1.0 mmol) was added.The mixture was stirred for 24h at room temperature, poured on water and extracted with DCM.The combined organic layers were dried over anhydrous magnesium sulfate and evaporated.Yields: 54% 1, 49% 9, 32% 10, 52% 11, 45% 12, 19% 13, 55% Method B. 42 Hydrazine hydrate (8.0 mmol) was added dropwise to the mixture of selenium powder (1.2 mmol) and lithium hydroxide (3.6 mmol) in DMF (3 mL).Reaction was heated to 120 o C and stirred for 15 min under argon atmosphere.After cooling to room temperature the amide (1.0 mmol) in DMF (2 mL) was added.

Table 1 .
Results of diselenides synthesis according to method C and D

Table 2 .
Results of the in vitro antioxidant activity measurement for benzisoselenazolones and diselenides