(4aR,5aR,11aR,12aS)-8-amino-2,12,12-trihydroxy-4a,5a,6,9,11,11a,12,12a-octahydro-[1,3,2]dioxaphosphinino[4',5':5,6]pyrano[3,2-g]pteridin-10(4H)-one2-oxide synthesis

Yield:-

Reaction Conditions:

Stage #1: (5aS,6R,7R,8R,9aR)-2-amino-6,7-dihydroxy-8-(hydroxymethyl)-3H,4H,5H,5aH,6H,7H,8H,9aH,10H-pyrano[3,2-g]pteridin-4-onewith dmap;di-tert-butyl dicarbonate in tetrahydrofuran at 50; for 20 h;Inert atmosphere;
Stage #2: with sodium methylate in methanol;

Steps:

1.B; 1.C; 1.D; 1.E; 1.F

B. Preparation of compounds 2 (a or b) and 3 (a, b or c).; Di-tert-butyl dicarbonate (10.33 g, 47.3 mmol) and DMAP (0.321 g, 2.63 mmol) were added to a stirred suspension of 1 (1.5 g, 5.26 mmol) in anhydrous THF (90 mL) at 50 °C under Ar. After 20 h a clear solution resulted. The solvent was evaporated and the residuechromatographed on silica gel (gradient of 0 to 40 % EtOAc in hexanes) to give two product fractions. The first product to elute was a yellow foam (1.46 g). The product was observed to be a mixture of two compounds by NMR containing mainly a product with seven Boc groups (2a or 2b). A sample was crystallized from EtOAc-hexanes to give 2a or 2b as a fine crystalline solid. MPt 189 - 191 °C. [a] *° -^3.6 (c 0.99, MeOH). 1H NMR (500 MHz, CDC1₃): δ 5.71 (t, J = 1.7 Hz, 1H), 5.15 (dt, 7= 3.5, - 1.0, 1H), 4.97 (t, J= 3.8, 1H), 4.35 (br t, J= ~ 1.7, 1H), 4.09- 3.97 (m, 3H), 3.91 (m, 1H), 1.55, 1.52, 1.51, 1.50, 1.45 (5s, 45H), 1.40 (s, 18H). ¹³C NMR (125.7 MHz, CDC1₃): δ 152.84 (C), 152.78 (C), 151.5 (C), 150.9 (C), 150.7 (2 ^χ C), 150.3 (C), 149.1 (C), 144.8 (C), 144.7 (C), 118.0 (C), 84.6 (C), 83.6 (C), 83.5 (C), 82.7 (3 ^χ C), 82.6 (C), 76.3 (CH), 73.0 (CH), 71.4 (CH), 67.2 (CH), 64.0 (C?), 51.4 (CH), 28.1 (CH₃), 27.8 (2 x CH₃), 27.7 (CH₃), 27.6 (3 x CH₃). MS-ESI+ for C₄₅H₇₂N₅0₁₉⁺, (M+H)⁺, Calcd. 986.4817 found986.4818. Anal, calcd. for C₄₅H₇iN₅0i₉ H₂0 54.39 C, 7.39 H, 6.34 N; found 54.66 C, 7.17 H, 7.05 N. A second fraction was obtained as a yellow foam (2.68 g) which by 1H NMR was a product with six Boc groups present (3a, 3b or 3c). A small amount was crystallized from EtOAc-hexanes to give colorless crystals, [a] ²_D° -47.6 (c, 1.17, CHC1₃). 1H NMR (500 MHz, CDC1₃): δ 1 1.10 (br s, exchanged D₂0, 1H), 5.58 (t, J= 1.8 Hz, 1H), 5.17 (d, J= 3.4 Hz, 1H), 4.97 (t, J= 3.9 Hz, 1H), 4.62 (s, exchanged D₂0, 1H), 4.16 (dd, J= 11.3, 5.9 Hz, 1H), 4.12 (dd, 7= 11.3, 6.4 Hz, 1H), 3.95 (dt, J= 6.1, 1.1 Hz, 1H), 3.76 (m, 1H), 1.51, 1.50, 1.49, 1.48, 1.46 (5s, 54H). ¹³C NMR (125.7 MHz, CDC1₃): δ 156.6 (C), 153.0 (C), 152.9 (C), 151.9 (C), 150.6 (C), 149.4 (2 x C), 136.2 (C), 131.8 (C), 1 16.9 (C), 85.0 (2 x C), 83.3 (C), 82.8 (C), 82.49 (C), 82.46 (C), 73.3 (CH), 71.5 (CH), 67.2 (CH), 64.5 (CH₂), 51.3 (CH), 28.0, 27.72, 27.68, 27.6 (4 x CH₃). MS-ESI+ for C₄₀H₆₄N₅Oi₇⁺, (M+H)⁺ calcd. 886.4287, found 886.4289.; C. Preparation of compound 4a, 4b or 4c.; Step 1 - The first fraction from B above containing mainly compounds 2a or 2b (1.46 g, 1.481 mmol) was dissolved in MeOH (29 mL) and sodium methoxide in MeOH (1M, 8.14 mL, 8.14 mmol) added. After leaving at ambient temperature for 20 h the solution was neutralized with Dowex 50WX8 (H⁺) resin then the solids filtered off and the solvent evaporated.Step 2 - The second fraction from B above containing mainly 3a, 3b or 3c (2.68 g, 3.02 mmol) was dissolved in MeOH (54 mL) and sodium methoxide in MeOH (1M, 12.10 mL, 12.10 mmol) added. After leaving at ambient temperature for 20 h the solution was neutralized with Dowex 50WX8 (H⁺) resin then the solids filtered off and the solvent evaporated.The products from step 1 and step 2 above were combined and chromatographed on silica gel (gradient of 0 to 15 % MeOH in CHC1₃) to give 4a, 4b or 4c as a cream colored solid (1.97 g). 1H NMR (500 MHz, DMSO d₆):6 12.67 (br s, exchanged D₂0, 1H), 5.48 (d, J= 5.2 Hz, exchanged D₂0, 1H), 5.43 (t, J= -1.9 Hz, after D₂0 exchange became a d, J= 1.9 Hz, 1H), 5.00 (br s, exchanged D₂0, 1H), 4.62 (d, J= 5.7 Hz, exchanged D₂0, 1H), 4.27 (d, J= 6.0 Hz, exchanged D₂0, 1), 3.89 (dt, J= 5.2, 3.8 Hz, after D₂0 became a t, J= 3.9 Hz, 1H), 3.62 (dd, J = 6.0, 3.7 Hz, after D₂0 exchange became a d, J= 3.7 Hz, 1H), 3.52 - 3.39 (m, 4H), 1.42 (s, 9H), 1.41 (s, 18H). ¹³C NMR (125.7 MHz, DMSO ?): δ 157.9 (C), 151.1, (C), 149.8 (2 ^χ C), 134.6 (C), 131.4 (C), 1 18.8 (C), 83.5 (2 ^χ C), 81.3 (C), 78.2 (CH), 76.5 (CH), 68.1 (CH), 66.8 (CH), 60.6 (CH₂), 54.4 (CH), 27.9 (CH₃), 27.6 (2 ^χ CH₃). MS-ESI+ for C25H40 5O1 j⁺, (M+H)⁺ calcd. 586.2719, found 586.2717.; D. Preparation of compound 5a, 5b or 5c; Compound 4a, 4b or 4c (992 mg, 1.69 mmol) was dissolved in anhydrous pyridine and concentrated. The residue was dissolved in anhydrous CH₂C1₂ (10 mL) and pyridine (5 mL) under a nitrogen atmosphere and the solution was cooled to -42 °C in an acetonitrile/dry ice bath. Methyl dichlorophosphate (187 μ, 1.86 mmol) was added dropwise and the mixture was stirred for 2 h 20 min. Water (10 mL) was added to the cold solution which was then removed from the cold bath and diluted with ethyl acetate (50 mL) and saturated NaCl solution (30 mL). The organic portion was separated and washed with saturated NaCl solution. The combined aqueous portions were extracted twice further with ethyl acetate and the combined organic portions were dried over MgS0₄ and concentrated. Purification by silica gel flash column chromatography (eluting with 2 - 20 % methanol in ethyl acetate) gave the cyclic methyl phosphate 5a, 5b or 5c (731 mg, 65%). 1H NMR (500 MHz, CDC1₃,):6 11.72 (bs, exchanged D₂0, 1H), 5.63 (t, J= 1.8 Hz, 1H), 5.41 (s, exchanged D₂0, 1H), 4.95 (d, J= 3.2 Hz, 1H), 4.70 (dt, J= 12.4, 1.8 Hz, 1H), 4.42 (dd, J= 22.1, 12.1 Hz, 1H). 4.15 (q, J= 3.7 Hz, 1H), 3.82 (s, 1H), 3.75 (s, 1H), 3.58 (d, J= 11.7 Hz, 3H), 2.10 (bs, exchanged D₂0, 1H + H₂0), 1.50 (s, 9H), 1.46 (s, 18H). ¹³C NMR (125.7 MHz, CDC1₃, centre line δ 77.0): δ 157.5 (C), 151.2 (C), 149.6 (2 x C), 134.5 (C), 132.3 (C), 117.6 (C), 84.7 (2 x C), 82.8 (C), 77.3 (CH), 74.8 (d, J= 4.1 Hz, CH), 69.7 (CH₂), 68.8 (d, J= 4.1 Hz, CH), 68.6 (d, J= 5.9 Hz, CH), 56.0 (d, J= 7.4 Hz, CH₃), 51.8 (CH), 28.1 (CH₃), 27.8 (CH₃). MS-ESI+ for C₂₆H₄₀N₅NaOi₃P⁺ (M+Na)⁺, calcd. 684.2252, found 684.2251.; E. Preparation of compound 6a, 6b or 6c; Compound 5a, 5b or 5c (223 mg, 0.34 mmol) was dissolved in anhydrous CH₂C1₂ (7 mL) under a nitrogen atmosphere. Anhydrous DMSO (104 ., 1.46 mmol) was added and the solution was cooled to -78 °C. Trifluoroacetic anhydride (104 xL, 0.74 mmol) was added dropwise and the mixture was stirred for 40 min. N,N-diisopropylethylamine (513 μ,,2.94 mmol) was added and the stirring was continued for 50 min at -78 °C. Saturated NaCl solution (20 mL) was added and the mixture removed from the cold bath and diluted with CH₂C1₂ (30 mL). Glacial acetic acid (170 iL, 8.75 mmol) was added and the mixture was stirred for 10 min. The layers were separated and the aqueous phase was washed with CH₂C1₂ (10 mL). The combined organic phases were washed with 5 % aqueous HC1, 3 : 1 saturated NaCl solution: 10 % NaHC0₃ solution and saturated NaCl solution successively, dried over MgS0₄, and concentrated to give compound 6a, 6b or 6c (228 mg, quant.) of suitable purity for further use. NMR (500 MHz, CDC1₃): δ 5.86 (m, 1 H), 5.07 (m, 1 H), 4.70 - 4.64 (m, 2 H), 4.49 - 4.40 (m, 1 H), 4.27 (m, 1 H), 3.56, m, 4 H), 1.49 (s, 9 H), 1.46 (s, 18 H) ppm. ¹³C NMR (500 MHz, CDC1₃): δ 157.5 (C), 151.1 (C), 150.6 (2 C), 134.6 (C), 132.7 (C), 1 16.6 (C), 92.0 (C), 84.6 (2 C), 83.6 (C), 78.0 (CH), 76.0 (CH), 70.4 (CH₂), 67.9 (CH), 56.2 (CH₃) 56.0 (CH), 28.2 (3 CH₃), 26.8 (6 CH₃) ppm.³¹P NMR (500 MHz, CDC1₃): δ - 6.3 ppm. 9; F. Preparation of compound 7: (4aR,5aR, llaR, 12aS)-l, 3, 2-Dioxaphosphorinof 4 ', 5 ': 5, 6Jpyrano[3, 2-gJpteridin- 10(4H)-one, 8-amino- 4a,5a, 6, 9, 11, 11a, 12, 12a-octahydro-2, 12, 12-trihydroxy-2-oxide; Compound 6a, 6b or 6c (10 mg, 14.8 μηιο) was dissolved in dry acetonitrile (0.2 mL) and cooled to 0 °C. Bromotrimethylsilane (19.2 μ, 148 μηιοΓ) was added dropwise and the mixture was allowed to warm to ambient temperature and stirred for 5 h during which time a precipitate formed. HCl_(aq) (10 μ, 37 %) was added and the mixture was stirred for a further 15 min. The mixture was centrifuged for 15 min (3000 g) and the resulting precipitate collected. Acetonitrile (0.5 mL) was added and the mixture was centrifuged for a further 15 min. The acetonitrile wash and centrifugation was repeated a further two times and the resulting solid was dried under high vacuum to give compound 7 (4 mg, 75 %). NMR (500 MHz, D₂0): δ 5.22 (d, J= 1.6 Hz, 1H), 4.34 (dt, J= 13, 1.6 Hz, 1H), 4.29 - 4.27 (m, 1H), 4.24 - 4.18 (m, lH), 3.94 (br m, 1H), 3.44 (t, J= 1.4 Hz, 1H). ³¹P NMR (500 MHz, D₂0): δ -4.8 MS-ESI+ forC₁₀H₁₅N₅O₈P⁺, (M+H)⁺ calcd. 364.0653, found 364.0652.

References:

WO2012/112922,2012,A1 Location in patent:Page/Page column 126-127; 129-132