Synthesis of some nucleosides derivatives from L- rhamnose with expected biological activity

Practical procedures for production of variously blocked compounds from L-rhamnose have been developed. These compounds are highly useful as indirect β-L-rhamnosyl donors. This approach represents a new method for the synthesis of aromatic nucleoside analogues and the synthesis of (3S, 4S, 5S, 6R) 3, 4, 5-triacetoxy-2-methyl-7,9-diaza-1-oxa-spiro [4,5]decane-10-one-8-thione (7).


Introduction
Rhamnose is a component of the outer cell membrane of acid-fast bacteria in the Mycobacterium genus, which includes the organism that causes tuberculosis [1][2][3]. Rhamnose has no role in mammalian metabolism so that compounds which interfere specifically with rhamnose metabolism should not have any deleterious effect on humans. It is possible that a chemotherapeutic approach to the treatment of diseases induced by mycobacteria, such as tuberculosis and leprosy, would be to find compounds which inhibit either the biosynthesis of Tdp-rhamnose or its subsequent incorporation into the cell wall. The term spironucleoside was introduced in 1990 to designate a class of spiranic sugar derivatives in which the anomeric carbon belongs to both the sugar ring and to a heterocyclic base [4][5][6].
Data on this type of compound were reported before 1990 but, as far as we are aware, without using the term spironucleoside. Of the different classes of nucleosides, the spironucleosides are probably the least well known. In the last eight years, other syntheses of hydantocidin [5], spirofuranoid derivatives of different heterocycles [7], pyranoid analogues of hydantocidin [8].
For the partial hydrolysis of the nitrile moiety in 4 HBr in acetic acid [10] afforded the corresponding (2, 3, 4-tri-O-acetyl-b-L-rhamnopyranose) formamide 5 after 3 h reaction time in 94% yield as an almost analytically pure crystalline product. The anomeric configuration of compounds 4 and 5 has been found to be β according to the measured coupling constants between H-1 and H-2 (J 1, 2~6 .3 Hz).
Photobromination of 5 with bromine in refluxing chloroform [11] gave 6 as an essentially pure product in an almost quantitative yield. Reaction of 6 with ammonium thiocyanate in nitromethane in the presence of elemental sulfur to suppress radical-mediated pathways [12] under nitrogen atmosphere gave spiro-thiohydantoin 7. The structures of the new heterocyclic derivatives have been unequivocally established by 1 HNMR spectroscopy.
2, 3-O-Isopropylidene-L-rhamnopyranose 8 was synthesized according to the reported procedure [13]. L-rhamnose 1 was reaction with dry acetone in the presence of p-toluene sulfonic acid to give 8. The 2, 3-O-isopropylidene group is used extensively as a blocking group in nucleosides synthesis as a means of enhancing the volatility of polar nucleosides. The reaction of 2, 3-O-isopropylidene-L-rhamnopyranose 8 with malononitrile in the presence of ammonium hydroxide in methanol afforded the corresponding 3-(2, 3-O-isopropylidene-b-L-rhamnopyranose) iminopropanenitrile 9 (Scheme 2).
The compound 9 underwent nucleophilic addition with benzylidenemalononitrile in refluxing ethanol in the presence of piperdine afforded the corresponding substituted pyridine derivative 11 in good yield. The structure of 11 was confirmed on the basis of elemental analysis and spectral data. The formation of 11 is assumed to occur via initial formation of the Michael addition of the amino group in 9 to activate the double bond in benzylidenemalononitrile followed by intramolecular cyclization, and then it loses hydrogen cyanide to afford the Pyridine derivative 11 [14][15][16].

Antibacterial activity
The antibacterial activities of the synthesized compounds were determined by the well diffusion method [17]. In this work, Escherichia coli (ATTC-25922), Klebsiella pneumoniae (ATCC 10031), Bacillus cereus (ATTC-10702), Salmonella typhimurium (ATTC-23564) were used to investigate the antibacterial activities. The prepared compounds were tested against the Gram positive bacteria (B. cereus) and Gram negative bacteria (E. coli). The bacterial liquid cultures were prepared in infusion broth for their activity tests. The compounds were dissolved in DMSO at concentration of 1 mg ml -1 . Antibacterial activity of DMSO against the test organisms was investigated, and was found to be nil. Approximately 1 cm 3 of a 24 h broth culture containing 10 6 cfu cm -3 was placed in sterile Petri dishes. Molten nutrient agar (15 cm 3) , kept at 45°C, was then poured into the Petri dishes and allowed to solidify. Six millimeter diameter holes were then punched carefully using a sterile cork borer and completely filled with the test solutions. The plates were incubated for 24 h at 37°C. After 24 h, the inhibition zone that appeared around the holes in each plate was measured. Antibacterial activity was determined by measuring the diameter of inhibition zone. Activity of each compound was compared with ciprofloxacin and sulphamethoxazol as standards [18,19].
These results are summarized in   10 had a considerable degree of inhibition against Bacillus Cereus and Klebsiella Pneumnoiae, compounds 9 and 10 had only weak inhibition against Escherichia coil EC.

Experimental Section
Melting points were determined with an Electro Thermal Mel-Temp II apparatus and are all uncorrected.
IR spectra were obtained in the solid state as potassium disc using a Perkin-Elmer model 1430 Spectrometer. 1 H NMR were recorded on aVarian/Gemini 200/MHZ spectrometer in DMSO-d 6 as a solvent and TMS as an internal standard (chemical shift in δ, ppm). Mass spectra were measured on an instrument "VG-7035" spectra were recorded at 70 or 15 electron volt. Elemental analysis was performed at the Micro analytical centre, Cairo University, Giza, Egypt.

2, 3-O-isopropylidene-L-rhamnopyranos (8)
The reaction mixture of L-rhamnose monohydrate (1 g; 5.5 mmol), dry acetone (50 mL), toluene-4-sulphonic acid monohydrate (100 mg) and 2, 2-dimethoxypropane (6.8 mL; 55 mmol) was stirred for 6 h. The reaction mixture was neutralized by the addition of sodium carbonate. The neutral mixture was filtered, washed with methanol and evaporated. A syrupy isopropylidene derivative was purified by column chromatography on silica gel. TLC indicated one major product 8 isolated as syrup.  A mixture of 9 (1.48 g, 5.48 mol) and benzylidenemalononitrile (0.54 g, 6 mol) in piperidene (10 mL) was refluxed for 3 h. The reaction mixture was then allowed to cool at room temperature. The solid product so formed was collected by filtration and recrystallized from ethanol as brown crystals, (71% yield  A mixture of 9 0.5 g (1.85 mol) was suspended in absolute ethanol and anhydrous pyridine (0.3 ml) and carbon disulphide 0.5 mol (0.6 g) were added slowly.

Conclusion
In conclusion, the preparation procedure follow in this work for synthesis of some nucleosides derivatives from L-rhamnose with expected biological activity show operation simplicity, cleaner reaction, easy work-up and improved yields. Spectroscopic and elemental analysis confirms the proposed structures of these compounds. The prepared compounds showed promising antibacterial activity against Gram-positive bacteria Bacillus cereus.