Synthesis of New Organoselenium Compounds Containing Nucleosides as Antioxidant LAILA

Selenium containing nucleosides derived from some heterocyclic moieties such as Pyridineselenol, and pyridazineselenol is described herein. Ribosylation of selenol compounds were prepared in good yield by silyation of selenol derivatives with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose followed by debenzoylation to afford the corresponding free N-nucleosides  and -1-(2,3,5-trihydroxy--D-ribofuranosyl)-2-seleno-4,6-dimethylpyridine-3-carbonitrile (6a,7a); b and a-1-(2,3,5-trihydroxy--D-ribofuranosyl)-3-seleno-5,6-diphenylpyridazine-4-carbonitrile (6b,7b). Newly synthesized compounds were characterized using the well known spectroscopic tools (IR, 1HNMR, 13CNMR and mass spectroscopy). Antioxidant activity of six selenonucleoside compounds (1a; 6a; 7a; 1b; 6b and 7b) was evaluated by animal assay model using experimental mice. The resulted data revealed that compounds 6a and 7b showed to be more active as antioxidant with a better performance of scavenging ability than the other compounds.

Stimulated by our recent work on the synthesis of selenium containing nucleoside analogues 2 , sulfa drugs 3 , and the synthesis of selenium containing amino acid analogues 4 , we decided to expand our interest to the introduction of an organoselenium compounds in the nucleoside framework and screened their biological activity as antioxidants.
The chemical structures of the nucleoside derivatives 4a, 4b, 5a, 5b, 6a, 6b, 7a and 7b were established and confirmed on the basis of their elemental analyses and spectral data (IR, 1 H and 13 C NMR) (see the Experimental section).
The IR spectra of compounds (4a, 5a, 4b and 5b) were observed at 2210 cm -1 due to CN group and stretching vibration frequencies of the benzoyl carbonyl groups C=O appeared at  1740, 1730, 1727 and 1724 cm -1 of compounds 4a, 5a, 4b and 5b respectively.In addition signals at 1625, 1620 cm -1 for the C=N group of compounds 4a, 5a, and at 1630 cm -1 of compounds 4b and 5b.
The IR spectra and the most important peaks for compounds (6a, 7a, 6b and 7b) were observed at  3400-3450 cm "1 due to (OH group) for compounds 6a and 6b respectively and signals at 3380 cm -1 due to (OH group) for compounds 7a and 7b.

Biological activity Toxicity studies
Toxicity parameters including LD 50 ; GPT

Fig. 1: Effects of synthesized compounds on activities of GPT and LDH enzymes
and LDH activities were determined and ranged in normal limits compared to infected untreated group with concentrations range up to 1000 mg kg-b.wt.The GPT, an enzyme which allows determining the liver function as indicator on liver cells damage and LDH enzyme is often used as a marker of tissue breakdown (Butt et al., 2002)  [6].

Antioxidant activity evaluation
Hepatic GSH-Rd and serum activities of SOD, GSH-S-transferase levels were measured as an indicator of antioxidant activity and result are present in Table 1.SOD and GSH-S-transferase are antioxidant enzymes that protect cells from oxidative stress of highly reactive free radicals and induces on the generation of free radicals in living cells.Result indicated that significant increasing (p < 0.05) in SOD, and GST activities in the treated groups at doses of 100 and 200 mg/kg compared to un-treated control group.No significant deference found between used doses (100 and 200 mg/kg) with all tested compounds.The highest SOD and GST, activities rather than GSH-Rd levels was monitored in animals treated with compounds 6a and 7b.(See Table 1, Fig. 1).

General
Melting points were determined by using the Kofler melting point apparatus, and were uncorrected.IR (KBr, cm -1 ) spectra were recorded on a Pye-Unicam SP3-100 instrument at Taif University.1H NMR spectra were obtained on a Varian (400 MHz) EM 390 USA instrument at King Abdel-Aziz University by using TMS as internal reference.13C NMR spectra were recorded on a JNM-LA spectrometer (100 MHz) at King Abdel-Aziz University, Saudi Arabia.Elemental analyses were obtained on an Elementar Vario EL 1150C analyzer.Mass spectra were recorded on a JEOL-JMS-AX 500 at Cairo National Research Center, Cairo, Egypt.Purity of the compounds was checked by thin layer chromatography (TLC) using silica gel plates.General Procedure.A mixture of 2-seleno-4,6-dimethylpyridine-3-carbonitrile or 3-seleno-5,6diphenylpyridazine-4-carbonitrile (1a,b) (0.02 mol) and hexamethyl di-silazane (20 ml) was heated under reflux for 24h with a catalytic amount of ammonium sulfate (0.01g).After that, the clear solution was cooled and evaporated till dryness to give the silyated derivative (2a,b), which directly was dissolved in 20 ml of dry 1,2-dichloroethane and then 1-O-acetyl-2,3,5-tri-O-benzoyl-Dribofuranose (3) (5.05 g, 0.01 mol) was added.The mixture was added dropwise onto a mixture of (10 ml trimethylsilyl trifluoromethanesulfonate (Triflate) in dry 1,2-dichloroethane (50 ml)).All mixture was stirred at room temperature for 24 h, and then washed with a saturated solution of aqueous sodium bicarbonate (3 × 50 ml), washed with water (3 × 50 ml), and dried over anhydrous sodium sulfate.The solvent was removed in vacuum and the residue was chromatographic on silica gel with chloroform: ethyl acetate (9: 1) as eluent to afford a white crystal from pure anomeric b and colorless crystals from a anomeric (4a,b and 5a,b) respectively.

Deprotection of 4 a,b and 5a, b. Synthesis of nucleosides 6a,b and 7a,b respectively. General Procedure
A mixture of each protected nucleoside 4a,b and 5a, b (0.001 mol for each), absolute methanol (20 ml) and sodium methoxide (0.055 g, 0.001mol) was stirred at room temperature for 48 h.The solvent was evaporated under vacuum to give a colorless solid, which was dissolved in hot water and neutralized with acetic acid.The precipitate compound was chromatographic on silica gel with chloroform: ethyl acetate (9: 1) as eluent to afford colorless and white crystals of the corresponding nucleosides 6 a,b and 7a,b respectively.

Biological experiments. Chemicals
Dimethyl sulfoxide (DMSO) and vitamin E were obtained from Sigma Chemical Co.(St.Louis, MO, USA).All enzymatic kits were purchased from Bioassays system, USA.

Experimental animals
Male Albino mice (20 ± 2 g) were obtained from department of animal science, cairo university and animals were handled under standard laboratory conditions with a 12-h light/dark cycle in a temperature of 25 ± C and a relative humidity of 55 ± 5 % controlled room.The basal diet used in these studies was certified feed to research laboratories animals.Food and water were available adlibitum.Cairo university animal care and use committee approved all protocols for the animal studies research.

Toxicity experiment
Male Albino mice of 6 animals per group and weighing between 25± 5 g were administered after overnight fasting with graded doses of (100-1000) mg kg -1 b.wt.intra peritoneal of each individual synthesized compounds suspended in DMSO.The toxicological effects were observed after 72 h of treatment in terms of mortality and expressed as LD 50 (Ghosh, 1984)  [8].Others biochemical parameters determined after 14 days of administration according to methods of Reitman and  Frankel (1957)  [9] for GPT activity and Bergmeyer (1974) [10]for LDH activity

Bioassays model design
The animals were randomly divided into fourteen groups of 6 mice each.The first group served as untreated normal control.Group 2 to Group 13 on 7th day, animals were pre-treated with individual synthesized compounds at 100 and 200 mg/ kg b, wt, per day p.o., respectively for 7 days.Group 14, animals were pre-treated with standard drug Vitamin E (100 mg/ kg b, wt, per day p.o) for 7 days.(Rai et al., 2006)  11 .
Twenty-four hours after the last administration, mice were sacrificed.Blood samples were collected and centrifuged at 4000×g at 4ae%C for 10 min for serums preparation.The liver was removed rapidly, washed and homogenized in icecold physiological saline to prepare 10% (w/v) homogenate.Then, the homogenate was centrifuged at 4000×g at 4ae%C for 10 min to remove cellular debris, and the supernatant was collected for biochemical analysis.

The biochemical assays. Measurement of Glutathione-S-Transferase activity (GST)
GST activity was determined as described by Habig et al., (1974)  12 .Reaction mixture containing 50 mM phosphate buffer, pH 7.5, 1 mM of 1-chloro-2, 4 dinitrobenzene (CDNB) and a appropriate volume of compound solution.The reaction was initiated by the addition of reduced glutathione GSH) and formation of S-(2, 4-dinitro phenyl) glutathione (DNP-GS) was monitored as an increase in absorbance at 334 nm.The result was expressed as µmol of CDNB conjugation formed /mg protein /min.

Measurement of Super Oxide Dismutase (SOD) activity
SOD activity was measured through the inhibition of hydroxylamine oxidation by the superoxide radicals generated in the xanthinexanthine oxidase system.(Kakkar et al. 1972)  [13].The results were expressed in units/mg protein.

Measurement of Glutathione Reduced (GSH-Rd) levels
GSH in liver and kidney tissues was determined according to the Ellman method (Ellaman, 1959)  14 , which measures the reduction of 5,50-dithio-bis (2-nitrobenzoic acid) (DTNB) (Ellman's reagent) by sulfhydryl groups to 2-nitro-5-mercaptobenzoic acid, which has an intense yellow color.The results were expressed in mg per g protein (mg/g protein).

Measurement of Protein content
Protein levels were determined spectrophotometrically at 595 nm, using comassie blue G 250 as a protein binding dye (Bradford,  1976)  15 .Bovine serum albumin (BSA) was used as a protein standard.showed to be more active as antioxidant with a better performance of scavenging ability than other compounds.The SOD activity of these molecules was compared with standard antioxidant (vitamin E).Selenonucleoside compounds are active sites of a large number of selenium dependent enzymes, such as antioxitant enzymes [Spallholz J.E 1994]  [7].The configuration structure of compounds 6a and 7b may more suitable for SOD and GST enzymes active center, so these compounds induce the antioxidants enzymes activity.