Synthesis of New Nucleoside Analogues From Benzimidazole and Evaluation of Their Antimicrobial Activity

Our goal in this research, some new nucleoside analogues was synthesized. Starting from ∝-D glucose which was converted to per acetylated β-D gluco pyronoside then converted to active from(1-Bromo Sugar (2) as a sugar moiety.The base moiety 2-substituted benzimidazole was prepared from condensation of phenylene diamine with different aromatic aldehydes, which were subjected to amino alkylation via Mannich reaction forming new nucleobase derivatives. Condensation of nucleobase with bromo sugar through nucleophilic substitution of anomeric carbon with nitrogen forming new protected nucleoside analogues then hydrolyzed with sodium methoxide in methanol to obtain our target, the free nucleoside analogues. All prepared compound were identified by FT-IR Spectroscopy and some of them with H 1 –NMR and C 13 -NMR Spectroscopy. The synthesized nucleoside analogues were screened for their antibacterial activity in vitro against four types of bacteria including, Bacillus Staphylococcus, aureus (Gram Positive), E.Coli and Pseudoman as (Gram Negative).Also were screened against four types of Fungi (Aspergines flurs, Aspergillus fumgntnts, Aspergillus niger and pencillum).


Introduction:
Nucleoside analogues have proven to be a highly successful class of anti-viral [1], anti-cancer,anti-tumerand chemotherpeutic agent [2].At the last decads, Nucleosides have sixmembered carbohydrate moiety have been evaluated for their potential antiviral [3], and antibiotic properties, also as building block in nucleic acid Synthesis [4].Nucleosides are large class of agents that include drugs for several diseases.The Synthesis and development of glycosidase inhibitors have been the focus of attention, because of their vital role played by carbohydrates invairety of biological processes.Benzimidazole ring is an important pharmaccophore in modern drug discover [5], compounds that exhibit functionality of benzimidazole and Its derivatives have been used in the area of pharmaceutical [6], these good and high profile applications of compound with benzimidazoles structures have prompted extensive studies for their method of synthesis.Modifications of the benzimidazole ring System that have made the studies of anthelmintic activity have provided [7].The most active drugs for anti-cancer, anticoagulant [8], anti-viral [9], anti-inflammatory, antihypertensive and anti-tumor [10].Based on the above significances, our research focus on synthesis of new nucleoside analogues containing 2substituted benzimidazole as anucleobase through Mannich base [11].

Materials and Methods:
The quality of all these chemical supplied from BDH England, and Fluga Merk,Pure materials used without purification.

Experimental instruments:
Melting points were recorded by Gallen Kamp, England, Melting point apparatus and were uncorrected.Infrared spectra were recorded using SHMADZU,FT-IR.8400(S) Spectrophotometer (Japan) as a thin film or KBr disk. 1 H-NMR and 13 C-NMR spectra were recorded with the help of ultra-high field 400 MHZ Avance III400 Bruker, Germany.At isfhon-University, Using Me 4 Si as the internal standard and DMSO-d 6 as a solvent, and was appeared at 2.5 PPM in 1 H-NMR AND 40.45 in 13 C-NMR spectrum.TLC plates were used with an aluminum backing (0.2mm, 60 F 254 ).
The acetylated sugar (0.380 g, 1.08 m mole) was dissolved in (3ml) of (50%) hydrogen bromide in glacial acetic acid which was added at (0 °C). the solution was kept at (0 °C) for one hour, and finally at room temperature for (15) min.washed with icewater (2x 15 ml) and then with saturated aqueous solutions of sodium bicarbonate to remove the remaining acid.After finial wash with icewater (20 ml), the organic phase was dried over MgSO 4 and solvent was removed to give compound (2) as a syrup.The isolated sugar bromide (2) was used directly for the nucleoside synthesis.General method for synthesis of 2-Substituted phenyl .1.H. benzimidazole (3-5) [13].
Compound structure and molecular formula M.p o C color yield Offwhite 78%

General procedure for hydrolysis of nucleoside analogues (20-26) [16]
A solutuin of (0.00026 mole) of the blocked nucleoside analogues in (7 ml) of (0.1m) methanolic sodium methoxide was refluxed with stirring for (0.5 h).The mixture was neutralized with acetic acid and evaporated to dryness.The aqueous phase evaporated to dryness under vacuum ,to obtain free nucleoside (20-26).

Biological activity
This test was performed by the disk diffusion method.Nutrient agar was added to (1L) of distilled water in suitable conical flask with stirring and heating unite complete dissolving then the disk was stoppered by cotton and the medium was sterilized in an autoclave for (20) minutes at (121) °C under pressure at (15) pound inch.The medium was placed in an-petridises about (20) mL for catch one was left to cool and solid filed.Agar plates was surface inculcated uniformly with 600 mL from both culture of tested microorganism.The impregnated disk were placed on the medium suitably spaced apart and the plates incubated at (5°C) for (1 h) to permit good diffusion and then transferred to an incubator at (37) °C for (24 h) for bacteria and (72 h) for fungi.The inhibition zones caused by virus compounds on the microorganisms were examined.

Results and Discussion:
The most common modification of nucleosides represent on important of medicinal compounds which have been found to behave as another agent and are currently used pharmaceuticals as antitumor, antiviral and antibiotics agents.Thus our target is to synthesize a new modified nucleoside analogues.Compounds (1 and 2) were confirmed by their physical properties due to the literature.

Table (5) FT-IR data cm -1 for compounds (3-26)
The FT-IR spectrum of compounds (1) showed several characteristic bands mainly the stretching band of carbonyl of acetyl group at 1744 cm -1 while compound (2) showed in addition to the carbonyl band the appearance of (C-Br) band at 780 cm -1.
On the other band benzimidazole is an important pharacophore due to its biological activities (18,19) , therefore, it was chosen as anucleobase, which was synthesized by condensation of phenylenediamine with substituted benzaldehyde using sodium with hydrogen sulfite as ring closing agent, according to the suggest mechanism showed in Scheme(2)
N(CH 3 ) 2 ,3-OH,4-OCH 3 R':1-butyl,2-butyl,1-naphtyl The synthetic rout was started with Dglucose as a sugar moiety and a new benzimidazole derivatives containing Mannich base as a base moiety, in a series of reaction steps.Scheme 1. D-glucose was protected with acetic anhydride in the presence of sodium acetate afforded B-D-glucose penta acetate (1) which was brominated using hydrogen bromide in glacial acetic to give acetylated sugar bromide (2).