5´,5´-Phosphodiesters and esterase labile triesters of 2´-C - methylribonucleosides

Bis(2´-C -methyladenosin-5´-yl) ( 11) , bis(2´-C -methylguanosin-5´-yl)


Introduction
Hepatitis C virus (HCV) infections constitute a worldwide health threat with an estimated 170 million infected individuals. 1,2Besides interferon based therapies, 3 inhibitors of the viral NS3-4A serine protease and the NS5B RNA polymerase have emerged as potential drug candidates. 4,5mong the RNA polymerase inhibitors, 2´-C-methylribonucleosides have received particular attention.2´-C-methyl-3´-O-(L-valinyl)cytidine (Valopicitabine) 6,7 and 4´-azido-3´,5´-di-O-(2methylpropanoyl)cytidine 8 showed antiviral activity in phase II and phase I clinal trials, respectively, and 2´-C-methyladenosine and 2´-C-methylguanosine inhibited HCV RNA replication in vitro. 9To terminate the synthesis of viral RNA, antiviral nucleoside analogues need to be converted to their 5´-triphosphates.Since conversion to the monophosphate often is the rate-limiting step, low antiviral activity may result from lack of this transformation by nucleoside kinases, and biodegradably protected nucleotides might, hence, be more potent drugs. 10For example, 2´-C-methylguanosine has been reported to suffer from inefficient uptake and phosphorylation. 11We now report on synthesis of novel type of pro-2´-Cmethylribonucleotides.Two 2´-C-methylribonucleosides are linked to each other via a 5´,5´phosphodiester bond that additionally bears an esterase labile protecting group, either a pivaloyloxymethyl or 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl group.The underlying idea is that intracellular carboxyesterases first releases the 5´,5´-phosphodiester, which is then cleaved by phosphodiesterases to nucleoside and nucleoside 5´-monophosphate.
The antiviral activity of the compounds will be tested by AliosBiopharma.

Experimental Section
General.Chemicals were purchased from Sigma-Aldrich, Fluka, Merck and Ramidus.MeCN, DCM, THF and pyridine were dried over 4Å molecular sieves and 1,4-dioxane over 3Å molecular sieves.Et3N was dried by refluxing over CaH2, distilled before use and stored over CaH2 lumps.Reactions were monitored with TLC (Merck silica gel 60 F254 aluminium sheets) or with 31 P-NMR.Column chromatography was performed on silica gel (Fluka silica gel 60 230-400 mesh). 1 H-, 13 C-, 31 P-and 2D-NMR spectra were recorded on Bruker Avance 400 or 500 spectrometer at 25ºC.The chemical shifts are given in ppm with reference to internal TMS, the coupling constants J are given in Hz.High resolution mass spectra were recorded on Bruker Daltonics microTOF-Q using electrospray ionization.

5´-O-(tert-Butyldimethylsilyl
. Levulinic acid (16.36 mmol; 1.90 g) was dissolved in dry 1,4-dioxane (20 mL) and cooled on an ice bath.Dicyclohexylcarbodiimide (DCC; 8.58 mmol; 1.77 g) was added portion wise within an hour.The reaction mixture was filtered and the precipitate was washed with dry 1,4-dioxane (10 mL).Compound 2 (3.16 mmol; 2.11 g) was coevaporated with dry pyridine and dissolved in the same solvent (10 mL) and the levulinic anhydride in 1,4-dioxane was added together with a catalytic amount of 4-dimethylaminopyridine (DMAP).The reaction mixture was stirred at r.t.overnight, heated on an oil bath at 54 °C for 5 hours and stirred again over night at r.t.The reaction mixture was evaporated to dryness, the residue was dissolved in DCM and washed with H2O, saturated aqueous NaHCO3 and brine.The crude product, dried over Na2SO4, was purified twice by silica gel chromatography, first eluting with 2% MeOH in DCM and then with 1% MeOH in DCM.According to the NMR spectrum, a mixture of products containing one or two levulinoyl groups was obtained, the singly acylated product predominating.The mixture of products was subjected to another levulinoylation.Levulinic acid (11.19 mmol; 1.30 g) was dissolved in dry DCM (20 mL) and DCC (5.48 mmol; 1.13 g) was added in one portion.The reaction mixture was stirred at r.t.overnight and filtered.The precipitate was washed with dry DCM (5 mL) and the DCM phases were combined.The filtrate and a catalytic amount of DMAP were added to the mixture (2.11 g) in dry DCM (5 mL).Owing to sluggish progress of the reaction, dry Et3N (0.50 mL) and some DMAP were after one day added to the reaction mixture.The reaction was completed in 4 days.The reaction mixture was washed with H2O, saturated aqueous NaHCO3 and brine, dried over Na2SO4, and evaporated to dryness.The crude product was purified on a silica gel column eluting with 1% MeOH in DCM.Compound 3 was obtained as yellowish solid in 72% yield (1.98 g). 1

N 2 -(4-Methoxytrityl)-3´-O-levulinoyl-2´-C-methylguanosine (6a).
Levulinic acid (4.82 mmol; 0.56 g) was dissolved in dry DCM (20 mL), DCC (2.90 mmol; 0.60 g) was added and the reaction mixture was stirred at r.t. for 4 hours.Precipitated dicyclohexylurea was removed by filtration and compound 5 (1.94 mmol; 1.33 g) in DCM (2 mL) and a catalytic amount of DMAP and Et3N (0.80 ml) were added.The reaction mixture was stirred at r.t.over night.The mixture was washed with H2O and brine.The organic layer was dried over Na2SO4 and evaporated to dryness.The crude product was purified on a silica gel column eluting with 1 to 3% MeOH in DCM.According to NMR spectroscopy, the main product bore only one levulinoyl group.The product was used as such for the next step.Accordingly, the levulinoylated nucleoside was dissolved in a mixture of dry THF (20 mL) and acetic acid (4 mL).Tetrabutylammonium fluoride hydrate (2.95 mmol, 0.77 g) was coevaporated from dry THF and added to the reaction mixture in the same solvent (1 mL).The mixture was evaporated to dryness after 21 hours stirring at r.t.The residue was equilibrated between EtOAc and saturated aqueous NaHCO3.The organic layer was washed with brine, dried over Na2SO4, and evaporated to dryness.The crude product was purified on a silica gel column eluting with 5% MeOH in DCM.The product was obtained as white solid in 50% yield (0.64 g). 1

2-Cyanoethyl bis[3´-O-levulinoyl-N 6 -(4-methoxytrityl)-2´-C-methylguanosin-5´-yl] phosphate (12).
Compound 6a (0.48 mmol; 0.32 g) dried over P 2 O 5 over night was dissolved in dry DCM (3 mL) under N2.Dry Et3N (2.41 mmol; 335 µL) and 1-chloro-1-(2-cyanoethoxy)-N,Ndiisopropylphosphinamine (0.53 mmol; 118 µL) were added.After 1 hour stirring at r.t., the mixture was passed through a short silica gel column eluting with EtOAc that contained 0.5% Et 3 N.The solvents were removed under reduced pressure and the residue was co-evaporated from dry MeCN.The residue was dissolved in dry MeCN (2 mL) and compound 6 (0.48 mmol; 0.32 g) and 1-H-tetrazole (0.96 mmol; 2.13 mL of 0.45 mol L -1 solution in MeCN) were added under N2, and the mixture was stirred for 1.5 hours.The phosphite ester obtained was oxidized as described above for compound 10.The phosphate triester 12 was isolated by conventional 5% aq NaHSO3/DCM workup.The crude product was purified on a silica gel column eluting with 5 to 10% MeOH in DCM.Compound 12 was obtained as white solid in 21% yield (0.15 g). 1  Bis(2´-C-methylguanosin-5´-yl) phosphate (13).Compound 12 (0.10 mmol; 0.15 g) was dissolved in 30% methanolic ammonia (25 mL) and the mixture was stirred at r.t.overnight, and evaporated to dryness under reduced pressure.The residue was dissolved in 80% aq AcOH (10 mL) and the mixture was stirred at r.t. for 3 days.The crude product obtained by evaporation to dryness was equilibrated between H2O and hexane.The aqueous phase was evaporated to dryness and the product was purified by HPLC as described above for compound 11.The product was dissolved in water and passed through an ion-exchange column (Dowex Na + -form, pivalate (POMCl, 25 µL, 0.18 mmol) was added and the reaction was allowed to proceed at 60 °C for 3 days.One equivalent of Et3N (13 µL, 0.09 mmol) and POMCl (13 µL, 0.09 mmol) was added and the reaction mixture was stirred for one more day.The mixture was equilibrated between water and EtOAc.The organic layer was washed with brine and evaporated to dryness.The residue was filtered through a short silica gel column eluting with 5% MeOH in DCM.The crude product was dissolved in DCM (600 µL) and hydrazinium acetate (0.044 g, 0.48 mmol) in MeOH (200 µL) was added.The reaction mixture was stirred at r.t. for 18 hours.The unreacted hydrazinium acetate was destroyed with acetone and the reaction mixture was evaporated to dryness.The residue was dissolved in 80% aqueous acetic acid (1 mL) and the mixture was stirred at r.t. for 24 hours.The crude product was purified by HPLC as described above for compound 11.Compound 20 was obtained as white solid in 15% yield (10 mg). 1