Non-natural nucleosides based on 1,2,4-triazolo[5,1-c ][1,2,4]triazin-4(6 H )-ones

Two regioselective methods for the synthesis of nucleosides in the series of 3-phenyl-and 3-ethoxycarbonyl-1,2,4-triazolo[5,1-c ][1,2,4]triazin-4-ones were developed. The first route involves a Vorbrüggen glycosylation reaction. The second one is based on condensation of 1,2,4-triazolo[5,1-c ][1,2,4]triazin-4-one sodium salts with protected 1-bromo-sugar derivatives.


Introduction
The synthesis of analogs of natural nucleosides based on modification of purines and pyrimidines is one of the most useful tools for development of antiviral compounds.In most cases, the structural transformations of nucleobases can be achieved by introduction or removal of different substituents.][3] Another strategy for synthesis of antiviral compounds is based on isosteres of natural nucleobases or heterocycles containing fragments of purines or pyrimidines.][6][7] Herein, we report the synthesis of abnormal nucleosides in the series of 1,2,4-triazolo[5,1c] [1,2,4]triazin-4-ones, considered as fused analogues of aza-isocytosines 8 and exhibiting antiviral activity. 9,10

Results and Discussion
The Vorbrüggen reaction is one of the widely used routes for the synthesis of nucleosides. 11This method of glycosylation involves interaction of protected sugar derivatives with appropriately silylated NH-heterocycles in the presence of a Lewis acid.We found that the conditions of the Vorbrüggen one-step method 12 were useful for glycosylation of 1,2,4-triazolo [5,1-c] [1,2,4]triazin-4-ones.Treatment on the NH-heterocycles 1a-e with N,Obis-(trimethylsilyl)acetamide (BSA) and trimethylsilyl triflate (TMSOTf) followed by addition of 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose at room temperature gave compounds 2a-e (Scheme 1).Although there are three possible positions for N-glycosylation (N1, N6 or N8), only products of N-1 glycosylation were observed.The sugar fragments in 2a-e had exclusively the βconfiguration.

Scheme 1
Removing the protecting acetyls of the compounds 2a-c in sodium methoxide solution gave nucleosides 3a-c.Meanwhile the deacetylation of 2d,e was carried out in acidic medium by mixture ethanol with acetyl chloride.Attempts to remove the acetyl-protecting groups in compound 2d,e by reaction with sodium ethoxide gave products of decomposition or incomplete deacetylation -for example, the monoacetyl derivative 4 was obtained from 2e.

Scheme 2
Following removal of the protection groups the nucleosides 6a-c were produced.The best conditions for deacetylation were found to be heating of 5a-c under reflux in MeONa/MeOH solution.
The signals in both the 1 H-and 13 C-NMR spectra of compounds 2a-e and 5a-c were assigned using 2D-1 H, 1 H COSY, 1 H, 13 C gHSQC and gHMBC experiments.The position of the tri-O-acetyl-β-D-ribofuranosyl and tetra-O-acetyl-β-D-glucopyranosyl fragments at the N-1 atom of the 1,2,4-triazine part in compounds 2a-e and 5a-c are evident from the observed cross-peaks between H-1' and C-8a in the HMBC spectra.NOESY spectra of 2a-e showed the β-configuration of the furanoses due to the presence of correlation of peaks Н-1' with H-4'.The structure of the acetylated nucleoside 2d was confirmed by X-ray diffraction (Fig. 1).It was found that the ribofuranosyl fragment of 2d has a 3'-exo twist conformation.
The derivatives of glucopyranose, 5a-c, have a β-configuration, and this was also confirmed by 2D-gNOESY experiments showing cross-peaks H-1' with H-3' and H-5', and vicinal coupling constant of H-1'-H-2' ( 3 J 9.0-9.5 Нz) in the 1 H NMR. 18 A single-crystal X-ray diffraction analysis was carried out in order to confirm the molecular structure of 5a-c.The molecular structure of 5c (Fig. 2) demonstrated that the glucopyranosyl fragment is attached at the azine part, and the sugar has the β-configuration.
The position of the protecting group in compound 4 has been determined by the 2D-HMBC spectrum, where the signal for the carbon of the acetyl group gave cross peaks with H-5'a and H-5'b.The structures of the nucleosides 3a-e and 6a-c were confirmed by 1 H NMR and mass spectra.

Experimental Section
General Procedures.IR spectra were recorded in KBr on a Perkin Elmer Spectrum One B FT-IR instrument.The 1 H-and 13 C-NMR spectra were measured on Bruker WM-250, Bruker DRX-400, and Bruker DRX-500 instruments.The 13 C-and 1 H-2D NMR spectra were recorded on the Bruker DRX-500 spectrometer in DMSO-d 6 .The mass spectra were obtained using a quadrupole Shimadzu LCMS-2010 system with a Supelco LC-18 column (4.6 × 250 mm), where a temperature of 60 °C was maintained.The mobile phase was acetonitrile (100 %).Positive chemical APCI ionization in the selective ion-monitoring (SIM) mode was used.The capillary voltage was set at 1.5 kV and cone voltage at 15.0 V. Microanalyses were performed on a Perkin Elmer PE 2400 series II CHNS/O analyzer.TLC was carried out on Silufol UV-254 plates using ethyl acetate as the eluent; spots were visualized by exposure to UV radiation.Column chromatography was performed on Merck Kieselgel-60.1-Bromo-2,3,5-tri-O-acetyl-α-Dglucose, 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose, β-D-glucose penta-acetate, N,O-bis-(trimethylsilyl)acetamide and trimethylsilyl triflate were purchased from Aldrich.RT denotes room temperature.

Table 1 .
Crystal date and structure refinement for compounds 2e and 5c