Synthesis and Biological Evaluation of Novel Pyrane Glycosides

A series of novel (5R)-5-((2S,3S)-3-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-3,6-dihydro-2H-pyran-2yl)-3-(4-fluorophenyl)-2,6-diphenyl-3,3a,5,6-tetrahydro-2H-pyrazolo[3,4-d]thiazoles 11a–g and (5R)-5-((2S,3S)3-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-3,6-dihydro-2H-pyran-2-yl)-3-(4-fluorophenyl)-6-phenyl-3,3a,5,6-tetrahydroisoxazolo[3,4-d]thiazoles 12a–g were synthesized by the reaction of chalcone derivatives of (R,Z)-2-((2S,3S)-3-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-3,6-dihydro-2H-pyran-2-yl)-5-(4-fluorobenzylidene)-3-phenylthiazolidin-4-ones 10a–g with phenylhydrazine and hydroxylamine hydrochloride. The chemical structures of newly synthesized compounds were elucidated by IR, NMR, MS and elemental analysis. The compounds 11a–g and 12a–g were evaluated for their antibacterial activity and antifungal activity.


Introduction
Carbohydrates, are the most ample class of bio-molecules, a vital source of energy and structural components having an important role in biological processes. Carbohydrates, besides being the most abundant class of bio-molecules, a vital source of energy and structural components, have an important role in biological processes, organic synthesis and chemical industries. 1 They have been mostly used in chemical industries and their large scale applications include their use as feedstocks in different chemical industries, like pharmaceutical, food, cosmetic and detergent industries. 2 In the formation of glycoconjugates (glycolipids, glycoproteins and polysaccharides) and in many biological processes they play decisive role in cell physiology such as intercellular recognition, bacterial and viral infection, cancer metastasis, apoptosis and neuronal proliferation, etc. 3 Their fascinating properties, such as hydrophilicity, lowered toxicity and emphasized bioactivities, in addition of carbohydrates affiliation to many systems make them often very effective. 4 Organic chemists have linked carbohydrates to various biologically potent compounds to enhance their biological applications, such as steroids, ami-noacids and other therapeutic agents. 5 One of the chemical reactions which is involved in making such links is carbohydrate affiliation with a potential compound through a triazole ring. 6 To obtain cyclised products with biological potential a process according to an efficient method is used, being an alkyne-azide cyclization reaction and the introduction of a triazole ring. 7 The strategy of linking a carbohydrate moiety with another species via a triazole ring is gaining importance in organic synthesis, natural products chemistry and biochemistry. 8 The combination of biocompatibility and presence of stereogenic centres stemming from the carbohydrate, together with the polar nature and possible hydrogen bonding ability of a triazole ring, makes gluco-based triazoles very fancinatining for organic synthetic chemists.

Results and Discussion
For the synthesis, the title compound was prepared according to the procedure outlined in the Scheme 1, where the key intermediate 8 is required. From 3,4,6-tri-O-acetyl-D-glucal (1) by treating with triethylsilane and boron trifluoride diethyl etherate, diacetyl-D-glucal (2) was prepared, giving with NaOMe in methanol at 0 °C after 1 h compound 3 (77%), which has on subsequent treatment with TBDMSCl in dichloromethane in the presence of NEt 3 after 12 h afforded TBS ether 4 (80%), which on treatment with propargyl bromide in toluene in the presence of tetrabutylammonium hydrogensulphate produced diether 5. After deprotection of TBS ether 5, the propargyl ether 6 was converted into triazole 7 (82%) by using 1,3-di-polar cycloaddition with para-chlorophenyl azide carried out at ambient temperature in the presence of CuSO 4 and sodium ascorbate in a mixture of 1:1 CH 2 Cl 2 -H 2 O. The synthesis of triazole-linked thiazolidinone glycosides was carried out by the condensation reaction of 8, obtained by the oxidation of 7 with IBX in acetonitrile. Compound 8 was in the next step reacted with R-substituted primary aromatic amine and thioglycolic acid in the presence of ZnCl 2 under microwave irradiation (Scheme 1) furnishing set of compound 9a-g. These compounds were isolated by conventional work-up, when the reaction was completed in only 5-10 minutes, the 9a-g were obtained in satisfactory yields. Then the compounds 9a-g were reacted with para-fluorobenzaldehyde in the presence of anhydrous NaOAc in glacial AcOH at reflux temperature gaving chalcone derivatives of triazole-linked thiazolidinone glycosides 10a-g. Further, these compounds upon cyclocondensation with arylhydrazines in the presence of anhydrous NaOAc in glacial AcOH at reflux temperature gave 11a-g in good yields. Compound 10a-g on cyclocondensation with hydroxylamine hydrochloride in the presence of anhydrous NaOAc in glacial AcOH at reflux temperature gave compounds 12a-g. By IR, NMR, and MS the structures of the synthesized compounds were confirmed and then evaluated for their antimicrobial activity.

Antimicrobial Activity
By the filter paper disc method, the antimicrobial activity of the synthesized compounds11a-g and 12a-g has been evaluated 44 against Staphylococcus aureus ATC-C6538P, Bacillus subtilis ATCC6633, Pseudomonas aeruginosa ATCC9027, and Echerichia coli ATCC8739. Antifun- gal activity of the synthesized compounds has been tested against Candida albicans ATCC2091, and Aspergillus niger, at a concentration of 500 μg/mL in DMF.
To culture the bacteria and fungi, nutrient agar and potato dextrose agars were used, respectively. The plates were cultured by the bacteria or fungi and incubated for 24 h at 37 °C for bacteria and for 72 h at 27 °C for fungi and then the inhibition zones of microbial growth surrounding the filter paper disc (5 mm) were measured in millimeters. Ampicillin and mycostatin, at a concentration 500 μg/mL, were used as standard against bacteria and fungi, respectively. All test results are shown in Table 1. From the data it is clear that compounds 11b, 12b, 11d, 12d, 11g, and 12g possess high activity, while compounds 11a, 12a, 11c, 11e, 12e, 11f, and 12f possess moderate activity against Gram positive bacteria. The compounds 11a, 12a, 11b, 12b, 11f, and 12f showed high activity as Gram negative microorganisms are concerned, while compounds 11c, 12c, 11g and 12g display moderate activity. Compounds 11e, 12e, 11g, and 12g also exerted high activity, while compounds 11a, 12a, 11c, 12c, 11d, 12d, 11b, and 12b have moderate activity against fungi.

Experimental
All the used reagents were supplied as commercially available. According to the literature, when necessary, the solvents used (except analytical reagent and grade) were dried and purified. By thin-layer chromatography (TLC) on pre-coated silica gel F254 plates from Merck the reaction progress and purity of the compounds was checked. They were is visualized either by exposure to UV light or dipping in 1% aqueous potassium permanganate solution. Silica gel chromatographic columns (60-120 mesh) were used for separations. Optical rotations were measured on an Perkin-Elmer 141 polarimeter by using a 2 mL cell with a path length of 1 dm with CHCl 3 or CDCl 3 as the solvent. By using Fisher-Johns apparatus all the melting points were measured and are corrected. IR spectra were recorded as KBr disks on a Perkin-Elmer FT IR spectrometer. Microwave reactions were carried out in mini lab microwave catalytic reactor (ZZKD, WBFY-201). On Varian Gemini spectrometer (300 MHz for 1 H and 75 MHz for 13 C) the 1 H NMR and 13 C NMR spectra were recorded; chemical shifts are reported as δ in ppm against TMS as the internal reference, coupling constants (J) are reported in Hz units. On a VG micro mass 7070H spectrometer mass spectra were recorded. Elemental analysis (C, H, N) were determined by a Perkin-Elmer 240 CHN elemental analyzer and were within ± 0.4% of theoretical values.

(2R,3S)-2-((tert-Butyldimethylsilyloxy)methyl)-3,6-dihydro-2H-pyran-3-ol (4)
At room temperature diacetate 2 (4.20 g, 19.53 mmol) was treated by a catalytic amount of sodium methoxide in 100 mL of methanol. The free hydroxyl unsaturated glycoside 3 was obtained in quantitative yield and used without further purification after evaporation of the solvent. This diol 3 was treated with 5 equiv of TBDMSCl (6.28 g, 42.28 mmol), 4.6 equiv of NEt 3 (6.4 mL, 44.8 mmol), and 0.10 equiv of imidazole (60 mg, 0.86 mmol) in CH 2 Cl 2 (60 mL) at room temperature for 24 h (until TLC analysis showed no more starting material). Thereafter, 25 mL of water were added and extraction with 3 × 30 mL of CH 2 Cl 2 followed; the organic layer was dried, evaporation of solvent under reduced pressure furnished the residue that was purified by column chromatography using petroleum ether/ethyl acetate as the eluent yielding the title compound 4 (3.84 g, 73.70%) as a colourless syrup. 1

General Procedure for the Synthesis of 11a-g
To the anhydrous sodium acetate (0.191 mmol) a mixture of compound 10a (0.191 mmol), phenyl hydrazine (0.191 mmol), in glacial acetic acid (10 mL), was refluxed for about 7 h. Then the reaction mixture was concentrated and cooled at room temperature, the solid was separated and filtered off, then it was washed thoroughly with water, the crude product was obtained and it was purified by column chromatography on silica gel with hexane-ethyl acetate as the eluent to afford pure compounds 11. (5R)-5-((2S,3S)-3- ((1-(4-Chlorophenyl)-1H-1,2,3 Similarly, all the compounds 11b-g were prepared according to the above procedure.

General Procedure for Synthesis of Compounds 12a-g
In anhydrous glacial acetic acid (10 mL), a mixture of compound 10a (0.191 mol), hydroxylamine hydrochloride (0.4 mol) and sodium acetate (0.191 mol) was refluxed for about 8 h. The reaction mixture was concentrated and then poured into ice cold water, the solid thus separated, was filtered, and washed with ice water, and crystallized from ethanol to afford pure 12a (87% yield) as a brown solid. Similarly all the compounds 12b-g prepared according to the above procedure.