Green Technique-Solvent Free Microwave Synthesis and Antimicrobial Evaluation of New Thiopyridine Arabinosides

A green protocol has been applied to synthesize a novel series of 3-cyano-2-(tri-O-acetyl-β-d-arabinopyranosylthio)pyridines in a short reaction time, in higher yields and with simpler operations, when compared with the conventional heating method. Deacetylation of the obtained acetylated arabinosides produced 2-(β-d-arabinopyranosylthio)-3-cyanopyridines. The structures of the obtained products were confirmed on the basis of spectroscopic data (FT-IR, 1D, 2D-NMR). The synthesized compounds were screened for the antimicrobial activity against a selection of Gram positive and Gram negative bacteria.


Introduction
In recent years, there has been increasing interest in the synthesis of nucleoside analogues due to their potential use to treat various diseases, such as AIDS, hepatitis, herpes, cancer and microbial infections [1][2][3][4][5][6][7]. On the other hand, thiopyridyl nucleosides have attracted noticeable attention because of their potential function as biological inhibitors, and ligands for carbohydrate-affinity chromatography of enzymes and proteins [8]. Nowadays, the development of green chemical methodologies is one of the most powerful tools in the synthesis of organic substances. Moreover solvent-free microwave irradiation approaches have been utilized to synthesize diverse chemical substances as an efficient green chemistry protocol. It has been reported to be an expeditious, simple, economical and environmentally benign synthetic methodology [9][10][11][12][13][14]. Thus, in continuation of our interest in developing green microwave syntheses of different novel functionalized pyridine compounds and their nucleosides with potentially significant medicinal and pharmacological applications due to their antagonistic activity against human cancer cells and antimicrobial activity [15][16][17], we were prompted to utilize a straightforward microwave-assisted route for the synthesis of thiopyridyl arabinosides as potential antimicrobial agents.

Chemistry
In the current report, we targeted dihydropyridine thioarabinosides 5a-j using new, simple and efficient procedures. Three different strategies were used (Scheme 1) and the resulting yields were
The structures of the obtained products 5a-j were confirmed on the basis of their elemental analyses and spectral data (LC-MS/MS, IR, UV, 1D-and 2D-NMR). Thus, the analytical data for 5d revealed a molecular formula C 26 H 28 N 4 O 7 S (LC-MS (ionization method): m/z 540 [M] + ). IR showed signals at υ 1655 and 2230 cm´1, assigned to the presence of a carbonitrile group. The 1 H-NMR spectrum of compound 5d showed a doublet at δ = 6.36 ppm with the spin-spin coupling constant J H-1"-H-2" = 2. 8 Hz. This small spin-spin coupling indicates the formation of the β-isomer in 4 C 1 and 1 C 4 . The use of piperidine as base to abstract hydrogen proton from the 2-thiopyridines 1a-j affords the thiopyridinium salts 2a-j which further attack the anomeric carbon of α-bromoarabinose 4 via equatorial attack. Inversion in the configuration of the anomeric carbon changes the stereochemistry of the obtained products 5a-j to the β-configuration through a S N 2 mechanism. The 13 C-NMR spectrum showed three signals at δ = 168.9, 170.1 and 170.8 ppm assigned to the acetoxycarbonyl sugar carbon. The 2D-NOESY spectrum showed that H-1" (δ = 6.36 ppm) had a cross-peak interaction with H-3" (δ = 5.39 ppm), while no cross peak interaction was observed between the anomeric proton H-1" and the methyl protons at C-6, indicating that the nucleosidic bond is formed between the anomeric carbon and the oxygen atom at C-2 forming an S-nucleoside as sole product. Dry ammonia or triethylamine in methanol were used to convert the protected nucleosides to their corresponding free nucleosides. The yield comparison between the two methods is given in Table 2. Table 1. Comparison between the microwave and conventional methods in the synthesis of 3-cyano2-(2",3",4"-tri-O-acetyl-β-D-arabinopyranosylthio) pyridines 5a-j.

Antimicrobial Activity
The antimicrobial activity of the synthesized compounds was investigated against a panel of standard Gram-negative (Proteus vulgaris and Escherichia coli) and Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacterial strains ( Figure 1). Concerning the antimicrobial activity data in Table 3, some of the synthesized compounds exhibited antibacterial potential comparable to reference drugs such as penicillin and ceftazidime. Concerning the activity against the Gram-positive bacterium Staphylococcus aureus, compounds 5j and 6j showed higher activity compared to the reference drugs, and compounds 5e, 6b and 6h exhibited good activity, whereas compounds 5c, 5h, 6c, 6e and 6f showed moderate activity. Compound 6e showed good activity against Gram-negative bacterium Escherichia coli. On the other hand, compound 6j showed good activity against Proteus vulgaris.   Table 3. Antimicrobial activity of all synthesized compounds 5a-j and 6a-j.

General Information
The microwave synthetic protocol was done using a CEM Microwave system (CEM Corporation, Matthews, VA, USA). Melting points were determined on a (Pyrex capillary) Gallenkamp apparatus (A. Gallenkamp & Co, London, UK). Infrared spectra were recorded with a Nicolet Nexus 470 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA) in the range 4000-400 cm´1 on samples in potassium bromide disks. 1 H-NMR, 13 C-NMR and 2D-NMR (COSY, NOESY, ROESY, G-HMBC and G-HMQC) spectra were obtained on a Gemini 400 MHz FT NMR spectrometer (Varian, Agilent Technologies, Edinburgh, UK) in CDCl 3 and DMSO-d 6 ; chemical shifts were recorded in δ (ppm) units, relative to Me 4 Si as an internal standard. The mass spectra were recorded on an LCMS-QP 800 LC-MS (Shimadzu, Tokyo, Japan). Elemental analysis data were obtained using a 2400 II series CHN Analyzer (Perkin Elmer, Waltham, MA, USA). Thin-layer chromatography (TLC) was carried out on precoated silica gel F254 plates (Merck, Kenilworth, NJ, USA) and UV light was used for visualization. Column chromatography was performed on a Merck silica gel. The reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification.

Microwave Method B
To a solution of 2-thiopyridine 1a-j (0.01 mol) and a catalytic amount of piperidine in acetone (5 mL), a solution of 2",3",4"-tri-O-acetyl-α-D-arabinopyranosyl bromide (4, 3.79 g, 0.11 mol) in acetone (5 mL) was added with stirring at room temperature. The mixture was irradiated for a suitable time as shown in Table 1 using the CEM Microwave system and then the solvent was removed under reduced pressure. Flash column was used to purify the product using n-hexanes/EtOAc (4:1) as eluent to afford the desired products 5a-j.

Method II
Dry ammonia gas was passed into a solution of protected nucleoside 5a-j (0.5 g) in dry methanol (20 mL) at 0˝C for 30 min. The reaction mixture was stirred until reaction completion as shown by TLC using chloroform/methanol 9/1 as eluent (4-6 h). The resulting mixture was then concentrated under reduced pressure to afford a crude solid. The crude products were purified by silica gel chromatography (chloroform/methanol, 9/1). The products were crystallized from methanol to furnish pure compounds 6a-j.