Synthesis of Some New Nucleoside Analogues Containing Seven Membered Ring and Studying Their Biological Activity

Abstract: In this work, a series of new Nucleoside analogues (D-galactopyranose linked to oxepanebenzimidazole moiety) was synthesized via multisteps synthesis. The first step involved preparation of two benzimidazoles 2styrylbenzimidazole and 2-(phenyl ethynyl) benzimidazole via reaction of phenylenediamine with cinnamic acid or β-phenyl propiolic acid. Electrophilic addition of the prepared benzimidazoles by three anhydrides in the second step afforded (4-6) and (14-16) which in turn were treated with 1,2,3,4-di-Oisopropylidene galactopyranose in the third step to afford a series of the desirable protected nucleoside analogues (7-9) ,(17-19)which after hydrolysis in methanolic sodium methoxidein the fourth step afforded the free nucleoside analogues (10-12) and (20-22) .The synthesized compounds were identified by FT-IR and some of them by 1 H-NMR and 13 C-NMR. The synthesized oxepane nucleoside analogues were screened for their antibacterial activity against three types of bacteria including Staphylococcusaureus ,Bacillus(gram positive) andE.coli (gram negative) bacteria repectively.


Introduction:
Oxepane diones that are heterocyclic compounds with seven-membered oxycyclic are found in a number of natural products such as zoapatanol ,regiolenyne and breveral [1].Nowaday they have been receiving more attention for their wide range of biomedical applications as cytotoxicity against cancer cell lines, antimicrobial activity and inhibition of several enzyme activities [2].Also they applied as achemotherapeutic used to treat advanced brain tumors [3], [4].On the other hand, benzimidazole has an important chemical entity in pharmaceuticals [5].Benzimidazole derivatives possess antibacterial effects, antifungal activity, HIV inhibitors, Open Access antiviral effects, antihypertensive agents, antiulcer activity, antiproliferative activity, antitumor activity, antioxidant agents [6].In another approach nucleoside analogues have found to be important moiety in creation of novel medical compounds.Even though, they have been studied a long time ago, there is still a great potential development of new therapeutic agents as antiviral compounds to treat diseases caused by HIV and hepatitis [7], antimicrobial agents [8], anticancer agents [9], [10].These observations inspire us to synthesis new nucleoside analogues containing oxepane and bezimidazole as a anucleobase and galactose as a asugar moiety which shows good biological activities against three types of bacteria.

Materials and Methods:
Melting points were recorded using Gallenkhamp electro thermal melting point apparatus.FT-IR spectra were recorded on SHIMADZU FT-IR 8400 fourier transform intrared spectrophotometer using KBr disc or thin films.'H-NMR spectra were recorded on Brukerspectrospin ultra shield magnet 300MHz instrument using Me 4 Si as the internal standard and DMSO-d6 as solvent.TLC plates were used with an aluminum backing (0.2mm, 60 F 254 ), the reactions were monitored by TLC and visualized by development of the TLC plates.Incubator Heraeus D-63450 (Germany) model was used for incubation samples for biological study.

1-Synthesis of 1,2:3,4-di-Oisopropylidene-D-galacto pyranose (1)
Zinc chloride (8.87g, 88mmol) was partially dissolved in (125ml) acetone and (0.4ml) concentrated sulfuric acid was added at room temperature to give a clear solution.(10g, 56mmol) D-galactosewas added in one portion and the resulting white suspension was stirred for 6 hours at room temperature.Asuspension of (20g ,189mmol) sodium carbonate in (30 ml) water added to the yellow reaction mixture at 0° C in medium sized portions.The suspension was allowed to stir for 30 min before filtration and solvent removal in vacuo to give the crude product as tallow oil below the aqueous layer.The organic fraction was separated from the aqueous layer, followed by further extraction with (3x50 ml) diethylether.The organic layer was dried over sodium sulfate [11], and the solvent removed in vacuo to yield 1,2:3,4-di-O-iso propylidene-Dgalactso (1) as a pale yellow syrup (87%),Rf=0.23 (Et 2 O).

7-Synthesis of 2-(phenyl ethynyl) benzimidazole (13)
The titled compound was prepared by following the same procedure used in preparation of 2cinnamoylbenzimidazole except using of β-phenyl propiolic acid instead of cinnamic acid.The product was purified by recrystallization from elhanol yield 73%.

11-Microbiological test:
Nutrient agar was added to (1L) of distilled water in suitable conical flask with stirring and heating until complete dissolving then the flask was stoppered by cotton and the medium was sterilized in an autoclave for 20 minutes at (121°) under pressure at 15 pound/inch.The medium was placed in petridishes about (20 ml) for each one and was left to cool and solidified.The studied bacteria and fungi were placed on the nutrient agar surface using the loop and by streaking processor then the discs saturated with tested compound solutions.The samples were incubated for 24 hours at 37°C [15].

Microbiological test
The prepared nucleosides are screened for their antibacterial activity against three types of bacteria Bacillu, Staphylococcusaureus (gram positive) and E.coli(gram negative) .They showed different biological activities against these bacteria as shown in Table (15).The result showed a high antibacterial activity of the prepared nucleosidesagaist gram negative more than gram positive, thus compounds (7)(8)(9)(10)(11)(12),(17-22) showed highantibacterial activity against E.coli.Also antibacterial activity of the prepared nucleosides depends on nature of substituents in their molecules, thus compounds (17),( 18) and (20) which are substituted phenyl ethynyl showed a high antibacterial activity against Staphylococcus aureusalso compounds (8) and ( 19) have a high antibacterial activity against this bacteria.Other compounds showed a moderate activity against this bacteria.Finally, compounds (7),( 18) and(21) showed a high antibacterial activity against Bacillus,compounds (9) ,(20) and( 22) showed no activity against this bacteria; other compounds showed a moderate activity against this bacteria.
2 , 3 ,4 -d i -o -i s o p r o p y li d e n e g a l a c t o p y r a n o s e ( 1 ) 6 -b r o m o -1 ,2 , 3 , 4 -d i -o -i s o p r o p y l id e n e g a l a c t o p y r a n o s e ( 2 ) + E t h a n o l 2 -s t y r y l b e n z im i d a z o le ( 3 ) , , 2 -b e n z i m i d a z o l y l-2 -p h e n y l o x e p a n e -4 , 7 -d i o n e ( 4 -6 ) ( 2 ) 6 -( N -( p h e n y l o x e p a n e -4 ,7 -d i o n e -3 -y l) b e n z i m id a z o l y l ) -1 ,2 , 3 , 4 -d i -o -i s o p r o p y l id e n e g a l a c t o p y r a n o s e ( 7 -9 ) .M e t h a n o l 6 -( N -( p h e n y l o x e p a n e -4 ,7 -d io n e -3 -y l ) b e n z i m i d a z o l y l ) g a l a c t o p y r a n o s e ( 1 0 -1 2 )