(2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a stirred solution of 1-deoxynojirimycin (100 g, 0.61 mol) in saturated aqueous sodium bicarbonate (1000 ml), benzyl chloroformate (95%, 121 g, 0.67 mol) was added dropwise at room temperature. After stirring at room temperature for 18 hr, the solution was extracted once with methylene chloride (300 ml) to remove any unreacted benzyl chloroformate. The aqueous layer was then extracted several times with ethyl acetate to give a total of 2.5-3 liters of the extract. The organic layer was then dried (Na2SO4), filtered and concentrated to give (2) a white solid (98 57 g, 54%), mp 101°-2° C., Anal calcd. for C14H19NO6C, 56.56, H, 6.44, N, 4.71 Found C, 56.33, H, 6.38, N, 4.58., 1H NMR (CD3OD) 7.2-7.4 (m, 5H), 5.15 (s, 2H), 4.23 (br m, 1H), 4.05 (br d., J=8 Hz, 1H), 3.87 (dd, J=6, 4 Hz, 1H), 3.78-3.85 (m, 2H), 3.70-3.78 (m, 2H), 3.45 (br d, J=8 Hz, 1H). the solution was extracted once with methylene chloride (300 ml) to remove any unreacted benzyl chloroformate The aqueous layer was then extracted several times with ethyl acetate to give a total of 2.5-3 liters of the extract The organic layer was then dried (Na2SO4) filtered concentrated

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The diol (17) (580 mg, 1.2 mmol) was stirred in glacial acetic acid (30 ml) in the presence of palladium black (100 mg) under hydrogen for 3 days, by which time no starting material (Rf 0.2) remained by t.l.c. (diethyl ether). Filtration and removal of the solvent then gave a gum which was purified by ion exchange chromatography with Dowex 50(H+) followed by Amberlite CG-400(Cl-) resins to give 1,5-dideoxy-1,5-imino-D-glucitol (3) (188 mg, 95%), which was crystallized as its hydrochloride salt. m.p. 204°-205° C. (lit.* 203°-206° C.). All spectroscopic data were identical to that of authentic material. --C NMR (D2O) free base δ: 79.0, 72.1, 71.1 (3d, C-2, C-3, C-4), 61.9 (t, C-6), 61.0 (d, C-5), 49.2 (t, C-1). Hydrogen chloride salt δ: 77.1, 68.6, 67.8 (3d, C-2, C-3, C-4), 60.8 (d, C-5), 58.5 (t, C-6), 46.7 (t, C-1). [*Fleet et al., Tetrahedron 43, 979 (1987)]. Filtration and removal of the solvent then gave a gum which was purified by ion exchange chromatography with Dowex 50(H+)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The diol (17) (580 mg, 1.2 mmol) was stirred in glacial acetic acid (30 ml) in the presence of palladium black (100 mg) under hydrogen for 3 days, by which time no starting material (Rf 0.2) remained by t.l.c. (diethyl ether). Filtration and removal of the solvent then gave a gum which was purified by ion exchange chromatography with Dowex 50(H+) followed by Amberlite CG-400(Cl-) resins to give 1,5-dideoxy-1,5-imino-D-glucitol (3) (188 mg, 95%), which was crystallized as its hydrochloride salt. m.p. 204°-205° C. (lit.* 203°-206° C.). All spectroscopic data were identical to that of authentic material. --C NMR (D2O) free base δ: 79.0, 72.1, 71.1 (3d, C-2, C-3, C-4), 61.9 (t, C-6), 61.0 (d, C-5), 49.2 (t, C-1). Hydrogen chloride salt δ: 77.1, 68.6, 67.8 (3d, C-2, C-3, C-4), 60.8 (d, C-5), 58.5 (t, C-6), 46.7 (t, C-1). [*Fleet et al., Tetrahedron 43, 979 (1987)]. Filtration and removal of the solvent then gave a gum which was purified by ion exchange chromatography with Dowex 50(H+)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1-deoxynojirimycin (DNJ) (1) was isolated from the roots of Morus alba (Moraceae) as described previously (28). 1-Deoxymannojirimycin (manno-DNJ) (2) and 1-deoxy-3,4-diepi-nojirimycin (gulo-DNJ) (5) have been recently isolated from the barks of Angylocalyx pynaertii (Leguminosae). 1-Deoxy-3-epi-nojirimycin (allo-DNJ) (3) was prepared by the microbial redox reaction at C-3 of the N-benzyloxycarbonyl derivative of DNJ as described previously (29) 1-Deoxygalactonojirimycin (DGJ) (4) and 1,2-dideoxy-galactonojirimycin (6) were prepared by the chemical epimerization of the 4-OH group of 1 and fagomine, respectively, according to the literature procedure (30). α-Homonojirimycin (α-HNJ) (7), α-homomannojirimycin (α-manno-HNJ) (8), and α-homoallonojirimycin (α-allo-HNJ) (9) were isolated from the whole plant of Aglaonema treubii (Araceae) as reported previously (31). α-Homogalactonojirimycin (α-galacto-HNJ) (10) was prepared from 2,3,4,6-tetra-O-benzyl-D-galactose by way of a Wittig chain extension and a mercuricyclization according to the literature procedure (33). N-Methyl-DGJ (11) was prepared by treatment of 4 with 37% HCHO and 80% formic acid according to the reference (34), and the N-ethyl (12), N-propyl (13), N-butyl (14) and N-hydroxyethyl (15) derivatives of 4 were prepared by treatment with the appropriate alkyl bromide and triethylamine in DMF. The reaction mixture of N-alkylation was evaporated in vacuo, and the residual syrup was resolved in MeOH and applied to an Amberlyst 15 column (H+ form), washed with MeOH, eluted with 0.5 M NH4OH, and concentrated. N-Alkylated derivatives were finally purified by Dowex 1x2 (OH− form) and Amberlite CG-50 (N4+ form) chromatography with water as eluent. β-1-C-Butyl-DGJ (16) was isolated from Adenophorae Radix as described previously (35). 4-epi-isofagomine (21), isofagomine (33) and its derivatives (34-37) were chemically synthesized as described previously (40). 2,5-Dideoxy-2,5-imino-D-mannitol (DMDP, 27), 1,4-dideoxy-1,4-imino-D-arabinitol (DAB, 28) were purified from Derris malaccensis and Morus alba, respectively (29). N-butyl-DNJ (30), N-dodecyl-DNJ (31), castanospermine (29) were from commercial sources. Calystegine A3 (17), calystegine A5 (22), calystegine B1 (23), calystegine B2 (18), calystegine B3 (24), calystegine B4 (25), and calystegine C1 (26) were prepared as published method (36, 40a), N-methyl calystegine A3 (19) and N-methyl calystegine B2 (20) were chemically synthesized.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1-deoxynojirimycin (DNJ) (1) was isolated from the roots of Morus alba (Moraceae) as described previously (28). 1-Deoxymannojirimycin (manno-DNJ) (2) and 1-deoxy-3,4-diepi-nojirimycin (gulo-DNJ) (5) have been recently isolated from the barks of Angylocalyx pynaertii (Leguminosae). 1-Deoxy-3-epi-nojirimycin (allo-DNJ) (3) was prepared by the microbial redox reaction at C-3 of the N-benzyloxycarbonyl derivative of DNJ as described previously (29) 1-Deoxygalactonojirimycin (DGJ) (4) and 1,2-dideoxy-galactonojirimycin (6) were prepared by the chemical epimerization of the 4-OH group of 1 and fagomine, respectively, according to the literature procedure (30). α-Homonojirimycin (α-HNJ) (7), α-homomannojirimycin (α-manno-HNJ) (8), and α-homoallonojirimycin (α-allo-HNJ) (9) were isolated from the whole plant of Aglaonema treubii (Araceae) as reported previously (31). α-Homogalactonojirimycin (α-galacto-HNJ) (10) was prepared from 2,3,4,6-tetra-O-benzyl-D-galactose by way of a Wittig chain extension and a mercuricyclization according to the literature procedure (33). N-Methyl-DGJ (11) was prepared by treatment of 4 with 37% HCHO and 80% formic acid according to the reference (34), and the N-ethyl (12), N-propyl (13), N-butyl (14) and N-hydroxyethyl (15) derivatives of 4 were prepared by treatment with the appropriate alkyl bromide and triethylamine in DMF. The reaction mixture of N-alkylation was evaporated in vacuo, and the residual syrup was resolved in MeOH and applied to an Amberlyst 15 column (H+ form), washed with MeOH, eluted with 0.5 M NH4OH, and concentrated. N-Alkylated derivatives were finally purified by Dowex 1x2 (OH− form) and Amberlite CG-50 (N4+ form) chromatography with water as eluent. β-1-C-Butyl-DGJ (16) was isolated from Adenophorae Radix as described previously (35). 4-epi-isofagomine (21), isofagomine (33) and its derivatives (34-37) were chemically synthesized as described previously (40). 2,5-Dideoxy-2,5-imino-D-mannitol (DMDP, 27), 1,4-dideoxy-1,4-imino-D-arabinitol (DAB, 28) were purified from Derris malaccensis and Morus alba, respectively (29). N-butyl-DNJ (30), N-dodecyl-DNJ (31), castanospermine (29) were from commercial sources. Calystegine A3 (17), calystegine A5 (22), calystegine B1 (23), calystegine B2 (18), calystegine B3 (24), calystegine B4 (25), and calystegine C1 (26) were prepared as published method (36, 40a), N-methyl calystegine A3 (19) and N-methyl calystegine B2 (20) were chemically synthesized.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Cells and viruses. BVDV-free MDBK cells (CCL 22) were obtained from the American Type Culture Collection and propagated in DMEM/F12 essential medium supplemented with penicillin (500 U/ml), streptomycin (500 U/ml), and 10% heat inactivated horse serum (Invitrogen). Cells were maintained in a humidified incubator at 37° C. with 5% CO2. BVDV (NADL strain). For infections, virus inoculum was added in complete medium and adsorbed for 1 hour at 37° C., the inoculum was then removed, the cells washed once with medium and fresh medium containing compounds added. Virus stocks were prepared by freeze-thawing the infected cells and culture supernatant three times followed by centrifugation at 1,000 g for 5 min. Stock titers were determined, and stocks were aliquoted and stored at −80° C. WNV was obtained from a cDNA clone of a human 2002 isolate from Texas; virus obtained from BHK cells electroporated with the in vitro synthesized RNA from this cDNA clone was passaged in Vero cells before use in antiviral assays. Dengue virus (DV) serotype 2 was a New Guinea C virus that had been passaged 28 times in suckling mouse brain, twice in Vero cells, and once in C6/36 mosquito cells. BVDV-free MDBK cells (CCL 22) were obtained from the American Type Culture Collection heat inactivated horse serum (Invitrogen) For infections, virus inoculum was added in complete medium the inoculum was then removed the cells washed once with medium and fresh medium containing compounds added Virus stocks were prepared by freeze-thawing the infected cells and culture supernatant three times followed by centrifugation at 1,000 g for 5 min stored at −80° C WNV was obtained from a cDNA clone of a human 2002 isolate from Texas

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A mixture of N-benzhydryl-1-deoxynojirimycin as prepared in Example 2 (Formula VI, 1.00 g, 3.06 mmol) and 20% PD(OH)2 /carbon (0.20 g) in 40 mL of methanol was hydrogenated (60 psig) in a Parr apparatus for 24 h. The mixture was filtered through Celite, and the filtrate was concentrated to a light grey foam. The foam was washed with hexane and then dissolved in water (10 mL) and stirred over Dowex® 50W-X8 ion exchange beads for 2 h. The beads were washed with 1N aqueous NH4OH solution until TLC analysis indicated the product had been completely removed from the resin. The combined ammonia washings were lyophilized to give 0.49 g (99%) of 1-deoxynojirimycin as a white foam, mp 198°-201° C. (literature mp 198°-202° C.: Kinast, G.; Schedel, M. Angew. Chem. Int. Ed. Eng. 1981, 20, 805). The 400-MHz 1H NMR spectrum of the material was identical to that of an authentic sample of 1-deoxynojirimycin (Sigma). for 24 h The mixture was filtered through Celite the filtrate was concentrated to a light grey foam The foam was washed with hexane dissolved in water (10 mL) The beads were washed with 1N aqueous NH4OH solution until TLC analysis had been completely removed from the resin

Conditions: temp:50.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 50 g (0.23 mol) of 6-amino-6-desoxy-L-sorbose hydrochloride were dissolved in 500 ml of distilled water, and the solution was added in the course of one hour to a solution of 11.2 g of dimethylaminoborane in 500 ml of distilled water, whilst stirring at a temperature of 50° C. The mixture was stirred for one hour at room temperature and one hour at 50° C., 5 ml of triethylamine were added to it, and it was then poured over a column containing 800 ml of strongly basic ion exchanger ("Lewatit" MP 500 OH- -form). The exchanger was washed with distilled water, and the runnings collected were concentrated to a syrup on a rotary evaporator. The concentrated syrup was crystallised at 50° C., with addition of a large amount of ethanol. The suspension of crystals was cooled and filtered off under suction, and the crystalline product was dried in a vacuum drying cabinet. The mixture was stirred for one hour at room temperature and one hour at 50° C. was then poured over a column containing 800 ml of strongly basic ion exchanger ("Lewatit" MP 500 OH- -form) The exchanger was washed with distilled water the runnings collected were concentrated to a syrup on a rotary evaporator The concentrated syrup was crystallised at 50° C., with addition of a large amount of ethanol The suspension of crystals was cooled filtered off under suction the crystalline product was dried in a vacuum drying cabinet

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The diol (17) (580 mg, 1.2 mmol) was stirred in glacial acetic acid (30 ml) in the presence of palladium black (100 mg) under hydrogen for 3 days, by which time no starting material (Rf 0.2) remained by t.l.c. (diethyl ether). Filtration and removal of the solvent then gave a gum which was purified by ion exchange chromatography with Dowex 50(H+) followed by Amberlite CC-400(Cl-) resins to give 1,5-dideoxy-1,5-imino-D-glucitol (3) (188 mg, 95%), which was crystallized as its hydrochloride salt. m.p. 204°-205° C. (lit.* 203°-206° C.). All spectroscopic data were identical to that of authentic material. 13C NMR (D2O) free base δ: 79.0, 72.1, 71.1 (3d, C-2, C-3, C-4), 61.9 (t, C-6), 61.0 (d, C-5), 49.2 (t, C-1). Hydrogen chloride salt δ: 77.1, 68.6, 67.8 (3d, C-2, C-3, C-4), 60.8 (d, C-5), 58.5 (t, C-6), 46.7 (t, C-1). [*Fleet et al., Tetrahedron 43, 979 (1987)]. Filtration and removal of the solvent then gave a gum which was purified by ion exchange chromatography with Dowex 50(H+)

Conditions: temp:50.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 50 g (0.23 mol) of 6-amino-6-desoxy-L-sorbose hydrochloride were dissolved in 500 ml of distilled water, and the solution was added in the course of one hour to a solution of 11.2 g of dimethylaminoborane in 500 ml of distilled water, whilst stirring at a temperature of 50° C. The mixture was stirred for one hour at room temperature and one hour at 50° C., 5 ml of triethylamine were added to it, and it was then poured over a column containing 800 ml of strongly basic ion exchanger ("Lewatit" MP 500 OH- -form). The exchanger was washed with distilled water, and the runnings collected were concentrated to a syrup on a rotary evaporator. The concentrated syrup was crystallised at 50° C., with addition of a large amount of ethanol. The suspension of crystals was cooled and filtered off under suction, and the crystalline product was dried in a vacuum drying cabinet. The mixture was stirred for one hour at room temperature and one hour at 50° C. was then poured over a column containing 800 ml of strongly basic ion exchanger ("Lewatit" MP 500 OH- -form) The exchanger was washed with distilled water the runnings collected were concentrated to a syrup on a rotary evaporator The concentrated syrup was crystallised at 50° C., with addition of a large amount of ethanol The suspension of crystals was cooled filtered off under suction the crystalline product was dried in a vacuum drying cabinet

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The diol (17) (580 mg, 1.2 mmol) was stirred in glacial acetic acid (30 ml) in the presence of palladium black (100 mg) under hydrogen for 3 days, by which time no starting material (Rf 0.2) remained by t.l.c. (diethyl ether). Filtration and removal of the solvent then gave a gum which was purified by ion exchange chromatography with Dowex 50(H+) followed by Amberlite CC-400(Cl-) resins to give 1,5-dideoxy-1,5-imino-D-glucitol (3) (188 mg, 95%), which was crystallized as its hydrochloride salt. m.p. 204°-205° C. (lit.* 203°-206° C.). All spectroscopic data were identical to that of authentic material. 13C NMR (D2O) free base δ: 79.0, 72.1, 71.1 (3d, C-2, C-3, C-4), 61.9 (t, C-6), 61.0 (d, C-5), 49.2 (t, C-1). Hydrogen chloride salt δ: 77.1, 68.6, 67.8 (3d, C-2, C-3, C-4), 60.8 (d, C-5), 58.5 (t, C-6), 46.7 (t, C-1). [*Fleet et al., Tetrahedron 43, 979 (1987)]. Filtration and removal of the solvent then gave a gum which was purified by ion exchange chromatography with Dowex 50(H+)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 2,3,6-Tri-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitol (0.2 g, 0.46 mmol) is dissolved in a 9:1 mixture of methanol and formic acid (10 ml) under an inert atmosphere and palladium 10% on charcoal (0.4 g) is added. The mixture is stirred overnight at room temperature. The catalyst is removed by filtration. The solvents are evaporated under reduced pressure. The residue is dissolved in methanol and the solution is filtered through a membrane (Millex-SR 0.5 μM). Evaporation of solvent gives a sticky solid which is further triturated in ethanol to give the expected 1,5-dideoxy-1,5-imino-D-glucitol as a beige powder (60 mg, 80%). is added The catalyst is removed by filtration The solvents are evaporated under reduced pressure The residue is dissolved in methanol the solution is filtered through a membrane (Millex-SR 0.5 μM) Evaporation of solvent gives a sticky solid which is further triturated in ethanol