Method and compositions for the synthesis of dioxolane nucleosides with beta-configuration

The present invention relates to methods and compositions for preparing biologically important nucleoside analogues containing 1,3-dioxolane sugar rings. In particular, this invention relates to the stereoselective synthesis of the beta (cis) isomer by glycosylating the base with an intermediate of formula (II) below a temperature of about −10° C. 1 wherein R 1 and L are as defined herein.

EXAMPLE 1a 
 2S-Benzyloxymethyl-4R-iodo-1,3 dioxolane and 2S-Benzyloxymethyl-4S-iodo-1,3 dioxolane (Compound &num;1) 11 A mixture consisting of 2S-benzyloxymethyl-4S acetoxy-1,3 dioxolane and 2S-benzyloxymethyl-4R-acetoxy-1,3 dioxolane in 1:2 ratio (6 g; 23.8 mmol) was dried by azeotropic distillation with toluene in vacuo. After removal of toluene, the residual oil was dissolved in dry dichloromethane (60 ml) and iodotrimethylsilane (3.55 ml; 1.05 eq) was added at −78° C., under vigorous stirring. The dry-ice/acetone bath was removed after addition and the mixture was allowed to warm up to room temperature (15 min.). The 1 H NMR indicated the formation of 2S-benzyloxymethyl-4R-iodo-1,3-dioxolane and 2S-benzyloxymethyl-4S-iodo-1,3 dioxolane. 1 H NMR (300 MHz, CDCl 3 ) &dgr;3.65-4.25 (2H,m); 4.50-4.75 (4H,m) 5.40-5.55 (1H, overlapping triplets); 6.60-6.85 (1H, d of d); 7.20-7.32 (5H,m). 
 EXAMPLE 1b 
 2S-Benzyloxymethyl-4R-iodo-1,3 dioxolane and 2S-Benzyloxymethyl-4S-iodo-1,3 dioxolane (Compound &num;1) 12 A mixture consisting of 2S-benzyloxymethyl-4S acetoxy-1,3 dioxolane and 2S-benzyloxymethyl-4R-acetoxy-1,3 dioxolane in 1:2 ratio (6 g; 23.8 mmol) was dried by azeotropic distillation with toluene in vacuo. After removal of toluene, the residual oil was dissolved in dry dichloromethane (60 ml) and diiodosilane (2.4 ml; 1.05 eq) was added at −78° C., under vigorous stirring. The dry-ice/acetone bath was removed after addition and the mixture was allowed to warm up to room temperature (15 min.). The 1 H NMR indicated the formation of 2S-benzyloxymethyl-4R-iodo-1,3-dioxolane and 2S-benzyloxymethyl-4S-iodo-1,3 dioxolane. 1 H NMR (300 MHz, CDCl 3 ) &dgr;3.65-4.25 (2H,m); 4.50-4.75 (4H,m) 5.40-5.55 (1H, overlapping triplets); 6.60-6.85 (1H, d of d); 7.20-7.32 (5H,m). 
 EXAMPLE 2 
 &bgr;-L-5′-Benzyl-2′-deoxy-3′-oxa-N-4-acetyl-cytidine (Compound &num;2) 13 The previously prepared iodo intermediate (example 1) in dichloromethane, was cooled down to −78° C. Persylilated N-acetyl cytosine (1.1 eq) formed by reflux in 1,1,1,3,3,3-hexamethyl disilazane (HMDS) and ammonium sulphate followed by evaporation of HMDS was dissolved in 30 ml of dichloromethane and was added to the iodo intermediate. The reaction mixture was maintained at −78° C. for 1.5 hours then poured onto aqueous sodium bicarbonate and extracted with dichloromethane (2×25 ml). The organic phase was dried over sodium sulphate, the solid was removed by filtration and the solvent was evaporated in vacuo to produce 8.1 g of a crude mixture. Based on 1 H NMR analysis, the &bgr;-L-5′-benzyl-2′-deoxy-3′-oxacytidine and its &agr;-L isomer were formed in a ratio of 5:1 respectively. This crude mixture was separated by chromatography on silica-gel (5% MeOH in EtOAc) to generate the pure &bgr;-L (cis) isomer (4.48 g). Alternatively, recrystallization of the mixture from ethanol produces 4.92 g of pure &bgr; isomer and 3.18 g of a mixture of &bgr; and &agr;-isomers in a ratio of 1:1. 1 H NMR (300 MHz, CDCl 3 ) &dgr;2.20 (3H,S,Ac); 3.87 (2H,m,H-5′), 4.25 (2H,m,H-2′); 4.65 (2H,dd,OCH 2 Ph); 5.18 (1H,t,H-4′); 6.23 (1H,m,H-1′); 7.12 (1H,d,H-5); 7.30-7.50 (5H,m,Ph); 8.45 (2H,m,NH&plus;H-6). 
 EXAMPLE 3 
 &bgr;-L-5′-Benzyloxy-2′-deoxy-3′-oxacytidine (Compound &num;3) 14 The protected &bgr;-L isomer (4.4 g) of example 2 was suspended in saturated methanolic ammonia (250 ml) and stirred at room temperature for 18 hours in a closed-vessel. The solvents were then removed in vacuo to afford the deacetylated nucleoside in pure form. 1 H NMR (300 MHz, CDCl 3 ) &dgr;3.85 (2H,m,H-5′); 4.20 (2H,m,H-2′); 4.65 (2H,dd,OCH 2 Ph); 5.18 (1H,t,H-4′); 5.43 (1H,d,H-5); 5.50-5.90 (2H,br.S,NH 2 ); 6.28 (1H,m,H-1′); 7.35-7.45 (5H,m,Ph); 7.95 (1H,d,H-6). 
 EXAMPLE 4 
 &bgr;-L-2′-deoxy-3′-oxacytidine (Compound &num;4) 15 &bgr;-L-5′-Benzyl-2′-deoxy-3′-oxacytidine from the previous example, was dissolved in EtOH (200 ml) followed by addition of cyclohexene (6 ml) and palladium oxide (0.8 g). The reaction mixture was refluxed for 7 hours then it was cooled and filtered to remove solids. The solvents were removed from the filtrate by vacuum distillation. The crude product was purified by flash chromatography on silica-gel (5% MeOH in EtOAc) to yield a white solid (2.33 g; 86% overall yield, &agr; D 22 &equals;−46.7° (c&equals;0.285; MeOH) m.p.&equals;192-194° C. 1 H NMR (300 MHz, DMSO- d 6 ) &dgr;3.63 (2H,dd,H-5′); 4.06 (2H,m,H-2′); 4.92 (1H,t,H-4′); 5.14 (1H,t,OH); 5.70 (1H,d,H-5); 6.16 (2H,dd,H-1′); 7.11-7.20 (2H,brS,NH 2 ); 7.80 (1H,d,H-6) 13 C NMR (75 MHz, DMSO- d 6 ) &dgr;59.5 (C-2′); 70.72 (C-5′); 81.34 (C-4′); 93.49 (C-1′); 104.49 (C-5); 140.35 (C-4); 156.12 (C-6); 165.43 (C-2). 
 EXAMPLE 5 
 &bgr;-L-5′-Benzyl-2′-deoxy-3′-oxa-5-fluoro-N4-acetyl-cytidine (Compound &num;5) 16 The previously prepared iodo derivatives (example 1) in dichloromethane, was cooled down to −78° C. Persylilated N-acetyl-5-fluorocytosine (1.05 eq) formed by reflux in 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and ammonium sulphate followed by evaporation of HMDS was dissolved in 20 ml of dichloromethane (DCM) and was added to the iodo intermediate. The reaction mixture was maintained at −78° C. for 1.5 hours then poured onto aqueous sodium bicarbonate and extracted with dichloromethane (2×25 ml). The organic phase was dried over sodium sulphate, the solid was removed by filtration and the solvent was evaporated in vacuo to produce 8.1 g of a crude mixture. Based on 1 H NMR analysis, the &bgr;-L-5′-benzyl-2′-deoxy-3′-oxa-5-fluoro-N4-acetyl-cytidine and its &agr;-L isomer were formed in a ratio of 5:1 respectively. This crude mixture was separated by chromatography on silica-gel (5% MeOH in EtOAc) to generate the pure &bgr;-L (cis) isomer (4.48 g). Alternatively, recrystallization of the mixture from ethanol produces 4.92 g of pure &bgr; isomer and 3.18 g of a mixture of &bgr; and &agr;-isomers in a ratio of 1:1. 1 H NMR (300 MHz, CDCl 3 ) &dgr;2.20 (3H,S,Ac); 3.87 (2H,m,H-5′), 4.25 (2H,m,H-2′); 4.65 (2H,dd,OCH 2 Ph); 5.18 (1H,t,H-4′); 6.23 (1H,m,H-1′); 7.12 (1H,d,H-5); 7.30-7.50 (5H,m,Ph); 8.45 (2H,m,NH&plus;H-6). 
 EXAMPLE 6 
 &bgr;-L-5′-Benzyl-2′-deoxy-3′-oxa-5-fluorocytidine (Compound &num;6) 17 The crude mixture from previous step (example 5) was suspended in methanolic ammonia (100 ml) and stirred for 18 hours at room temperature in a closed reaction vessel. The solvents were removed in vacuo to afford the deacetylated mixture which was separated by flash chromatography on silica gel (2% to 3% MeOH in EtOAc) to yield 1.21 g pure &bgr; isomer (yield 45% with respect to this isomer). 
 EXAMPLE 7 
 &bgr;-L-2′-deoxy-3′-oxa-5-fluorocytidine (Compound &num;7) 18 The deacetylated pure &bgr;-L isomer (900 mg; 2.8 mmol) prepared as described in example 6 was dissolved in EtOH (40 ml) followed by addition of cyclohexene (3 ml) and palladium oxide catalyst (180 mg). The reaction was refluxed for 24 hours and the catalyst was removed by filtration. The solvents were removed from the filtrate by vacuum distillation. The crude product was purified by flash chromatography on silica-gel (5% to 7% MeOH in EtOAc) to yield a white solid (530 mg; 82% yield). (&agr; 22 D )&equals;−44.18° (c&equals;0.98; MeOH). 1 H NMR (300 MHz, DMSO-d 6 ); &dgr;3.62-3.71 (2H,m,H-5′); 4.03-4.13 (2H;m,H-2′); 4.91 (1H,t,H-4′); 5.32 (1H,t,OH); 6.11 (1H;t;H-1′); 7.53-7.79 (2H,b,NH 2 ); 8.16 (1H;d,H-6); 13 C NMR (75 MHz, DMSO-d 6 ); &dgr;59.34 (C-2′); 70.68 (C-5′); 80.78 (C-4′); 104.53-(C-1′); 124.90, 125.22 (C-4); 134.33, 136.73 (C-5); 153.04 (C-2); 156.96, 157.09 (C-6). 
 EXAMPLE 8 
 Isomeric Purity Determination of &bgr;-L-2′-deoxy-3′-oxacytidine Nucleoside Analogues The determination of the isomeric purity (&bgr;-L versus &agr;-L and &bgr;-L versus &bgr;-D isomers) was determined on a Waters HPLC system consisting of a 600 controller pump for solvent delivery, 486 uv detector, 412 WISP auto sampler and a 740 Waters integrator module. An analytical chiral reverse phase cyclobond I RSP column (Astec, 4.6×250 mm i.d.) was used and packed by the manufacturer with &bgr;-cyclodextrin derivatized with R′S-hydroxypropyl ether. The mobile phase consisted of acetonitrile (A) and water containing 0.05% triethylamine (B) with the pH adjusted to 7.05 by glacial acetic acid. The column was operated under isocratic conditions at 0° C. using a mixture of 5% A and 95% B. Such conditions are modifications of those reported in DiMarco et al. ( J. Chromatography, 1993, 645, 107-114). The flow rate was 0.22 ml/min and the pressure was maintained at 648-660 psi. Detection of nucleosides was monitored by uv absorption at 215 and 265 nm. Samples of &bgr;-D isomer and racemic compounds were prepared as reported (Belleau et al. Tetrahedron Lett 1992, 33, 6948-6952) and used for internal references and co-injection. Under these conditions the isomeric purity of compound &num;4 produced according to example 4 was >99% and that of compound &num;7 according to example 7, was >96%. The isomeric purity of dioxolane nucleosides having been prepared according to the general scheme 2, under varying conditions i.e. temperature and Lewis acid is represented in table 1 below. Those prepared at temperatures above −10° C. exhibited reduced stereoselectivity. 19 1 TABLE 1 Base Lewis acid Temperature(° c.) Cis:trans 5F-N(Ac)-cytosine TMSI a:-78 b:-78 8:1 5F-N(Ac)-cytosine SiH 2 I 2 a:-78 b:-78 7:2 N(Ac)-cytosine TMSI a:-78 b:-78 5:1 note: all reactions in DCM solvent and bases silylated with HMDS.