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DOI: 10.1055/s-0042-1751507
A Facile Synthesis of 3′-Fluoro Hexitol Adenosine and Guanosine Phosphoramidites
We thank Ionis Pharmaceuticals Inc. for financial support.
Dedicated to Prof. Har Gobind Khorana
Abstract
We report a convenient and scalable synthetic approach for the synthesis of 3′-fluoro hexitol adenosine and guanosine nucleoside analogues and corresponding phosphoramidites in good yield. 1,5-Anhydro-4,6-O-benzylidene-d-glucitol was converted into 1,5-anhydro-4,6-O-benzylidene-3-deoxy-3-fluoro-2-O-trifluoromethanesulfonyl-d-altritol in a three-step process. Glycosylation using adenosine or 2-amino-6-iodopurine yielded the corresponding nucleoside analogues in excellent yield. Based on this strategy, a highly concise and scalable synthesis of 3′-fluoro hexitol purine nucleosides (1–2 g, 18–21% overall yield) was accomplished, which will enable the use of 3′-fluoro hexitol nucleic acids for genetic medicine development and diagnostic applications.
Key words
antisense oligonucleotides - FHNA adenosine nucleoside - FHNA guanosine nucleoside - hexitol nucleic acids - chemically modified siRNAsSupporting Information
- Supporting information for this article is available online at https://doi.org/10.1055/s-0042-1751507.
- Supporting Information
Publication History
Received: 23 July 2023
Accepted after revision: 19 September 2023
Article published online:
19 October 2023
© 2023. Thieme. All rights reserved
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References and Notes
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- 13 1,5-Anhydro-4,6-O-benzylidene-3-deoxy-3-fluoro-2-O-pivaloyl-d-altritol (6). A solution of trifluoromethanesulfonic anhydride (6.0 mL, 35.6 mmol) in dichloromethane (6 mL) was added to a cold (0 °C) solution of 4 (10.0 g, 29.7 mmol) in dichloromethane (45 mL) and pyridine (4.7 mL, 58.5 mmol). After stirring for 1 h the reaction was quenched by adding water and the organic layer was washed with water (100 mL) and brine (100 mL), then dried (Na2SO4) and concentrated to provide the 3-O-trifluoromethyl derivative, which was used without further purification. 1H NMR (300 MHz, CDCl3): δ = 7.53–7.42 (m, 2 H), 7.42–7.32 (m, 3 H), 5.59 (s, 1 H), 5.10 (s, 2 H), 4.48–4.33 (m, 1 H), 4.32–4.15 (m, 1 H), 3.90–3.69 (m, 2 H), 3.57–3.42 (m, 1 H), 3.40–3.22 (m, 1 H), 1.24 (s, 9 H). A solution of 3-O-trifluoromethanesulfonyl derivative from above and cesium fluoride (16.7 g, 109.9 mmol) in tert-butanol (110 mL) was heated at 70 ° for 2 h. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (200 mL) and the organic layer was washed with water (200 mL) and brine (220 mL), then dried (Na2SO4) and concentrated. Purification by column chromatography (silica gel, eluting with 10 to 20% ethyl acetate in hexanes) provided compound 6 (9.4 g, 94%). 1H NMR (300 MHz, CDCl3): δ = 7.49 (m, 2 H), 7.37 (m, 3 H), 5.56 (s, 1 H), 5.29–5.02 (m, 1 H), 5.02–4.81 (m, 1 H), 4.49–4.32 (m, 1 H), 4.22–4.04 (m, 1 H), 3.99–3.54 (m, 7 H), 1.23 (s, 9 H). 19F NMR (282 MHz, CDCl3): δ = –217. 53 (m, H-F coupled). HRMS (ESI): m/z [M + H]+ calcd for C18H24FO5: 339.1608; found: 339.1609.
- 14 1,5-Anhydro-4,6-O-benzylidene-3-deoxy-3-fluoro-d-altritol (7). Potassium carbonate (9.9 g, 71.6 mmol) was added to a solution of compound 6 (9.0 g, 26.6 mmol) in methanol (100 mL). After stirring at room temperature for 3 h, the solvent was evaporated under reduced pressure and the residue was partitioned between ethyl acetate (100 mL) and water (100 mL). The organic layer was dried (Na2SO4) and concentrated to yield 7 (6.5 g, 96%), which was used without further purification. 1H NMR (300 MHz, CDCl3): δ = 7.58–7.30 (m, 5 H), 5.54 (s, 1 H), 5.23–4.94 (m, 1 H), 4.39 (dd, J = 4.7, 10.0 Hz, 1 H), 4.02–3.43 (m, 6 H), 2.25–2.08 (m, 1 H). 13C NMR (75.47 MHz, CDCl3): δ = 136.8, 129.3, 128.4, 126.3, 102.2, 90.5, 88.1, 77.7, 69.2, 66.9, 65.8. 19F NMR (282 MHz, DMSO-d6): δ = –217. 53 (m, H-F coupled). MS (ESI): m/z [M + H]+ calcd for C13H16FO4: 254.26; found: 254.16.
- 15 1,5-Anhydro-4,6-O-benzylidene-3-deoxy-3-fluoro-2-O-trifluoromethanesulfonyl-d-altritol (8). Trifluoromethanesulfonic anhydride (5.2 mL, 30.8 mmol) was added to a cold (0 °C) solution of compound 7 (6.5 g, 25.7 mmol) in dichloromethane (39 mL) and pyridine (6.1 mL, 75.9 mmol). After stirring for 1 h, the reaction was quenched by adding water (10 mL) and the organic layer was washed with water (50 mL) and brine (50 mL), then dried (Na2SO4) and concentrated to provide crude 8 (9.4 g, 95%), which was used without further purification. 1H NMR (300 MHz, CDCl3): δ = 7.58–7.32 (m, 5 H), 5.55 (s, 1 H), 5.28 (1 H, d, J = 55 Hz), 5.02–4.85 (m, 1 H), 4.42 (dd, J = 4.9, 10.4 Hz, 1 H), 4.09 (dd, J = 5.7, 10.8 Hz, 1 H), 4.01–3.80 (m, 2 H), 3.78–3.50 (m, 2 H). 13C NMR (75 MHz, CDCl3): δ = 136.4, 129.5, 128.4, 126.2, 102.3, 87.5, 85.0, 78.4, 78.1, 77.1, 76.9, 68.8, 66.1, 66.0, 63.5. 19F NMR (282 MHz, CDCl3): δ = –74.77 (s, 3 F), –215. 96 (m, 1 F, H-F coupled). MS (ESI): m/z [M + H]+ calcd for C14H15F4O6S: 387.04; found: 387.01
- 16 1,5-Anhydro-4,6-O-benzylidene-2,3-dideoxy-3-fluoro-2-(N-6-benzoyl-adenin-9-yl)-d-altritol (13). Compound 12 (3.9 g 10.5 mmol) was dissolved in anhydrous pyridine (50 mL), then cooled to 0 °C and the solution was treated with benzoyl chloride (1.97 mL, 16.9 mmol). Stirring was continued at 0 °C for 15 min at which time the mixture was warmed to room temperature over 2.5 h. The mixture was cooled to 0 °C, quenched with H2O (20 mL) and stirred for 15 min. Concentrated aqueous NH4OH (20 mL) was added to the mixture with stirring for 30 min. The mixture was concentrated under reduced pressure to approximately 40 mL and poured into ethyl acetate (200 mL). The mixture was washed with saturated aqueous NaCl (3 × 500 mL), dried (Na2SO4), filtered, and evaporated. The residue was purified by silica gel chromatography (methanol/dichloromethane, 1–5%) to yield 13 (3.6 g, 72%). 1H NMR (300 MHz, CDCl3): δ = 9.23 (s, 1 H), 8.77 (s, 1 H), 8.55 (s, 1 H), 8.03 (d, J = 9.0 Hz, 2 H), 7.68–7.28 (m, 8 H), 5.51 (s, 1 H), 5.31 (d, J = 48.0 Hz, 1 H), 5.04 (br. s, 1 H), 4.59–4.35 (m, 3 H), 4.21–4.07 (m, 1 H), 3.86–3.59 (m, 2 H). 13C NMR (75 MHz, CDCl3): δ = 164.8, 152.9, 152.3, 149.8, 141.4, 136.5, 133.3, 132.9, 129.3, 128.8, 128.3, 128.0, 126.1, 122.7, 102.6, 85.8, 83.4, 75.2, 75.9, 68.8, 67.6, 65.0, 53.7, 53.3. 19F NMR (282 MHz, CDCl3): δ = –200.8 (m, H-F coupled). MS (ESI): m/z [M + H]+ calcd for C25H23FN5O4: 476.17; found: 476.02.
- 17a 1,5-Anhydro-(2-amino-6-iodopurin-9-yl)-4,6-O-benzylidene-2,3-dideoxy-3-fluoro-2-d-altritol (17). Compound 8 (2.9 g, 7.51 mmol) and 6-iodo-2-aminopurine tetrabutylammonium salt 16 (8.5 g, 17.6 mmol, prepared as described in ref. 17b), were dissolved in anhydrous hexamethylphosphoramide (HMPA, 26 mL). After stirring at room temperature for 18 h, the reaction mixture was poured into ethyl acetate (100 mL) and washed with water (100 mL) and brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/hexanes, 50%) to yield 17 (2.8, 75%). 1H NMR (300 MHz, DMSO-d 6): δ = 8.17 (s, 1 H), 7.36 (s, 5 H), 7.00 (s, 2 H), 5.68 (s, 1 H), 4.99–5.31 (d, J = 48 Hz, 1 H), 4.70–4.84 (m, 1 H), 4.35–4.53 (m, 1 H), 4.14–4.35 (m, 2 H), 3.56–4.10 (m, 3 H). 13C NMR (75.47 MHz, CDCl3): δ = 159.0, 149.8, 139.7, 13654, 131.7, 129.3, 128.3, 126.1, 123.0, 102.6, 85.6, 83.1, 75.2, 74.9, 68.8, 67.5, 64.8, 53.5, 53.1. 19F NMR (282 MHz, DMSO-d 6): δ = –201.8 (m, H-F coupled). MS (ESI): m/z [M + H]+ calcd for C18H18FIN5O3: 498.27; found: 498.20.
- 17b Bisacchi GS, Singh J, Godfrey JD. Jr, Kissick TP, Mitt T, Malley MF, Di Marco JD, Gougoutas JZ, Mueller RH, Zahler R. J. Org. Chem. 1995; 60: 2902
- 18 1,5-Anhydro-4,6-O-benzylidene-2,3-dideoxy-3-fluoro-2-(guanin-9-yl)-d-altritol (18). Compound 17 (2.7 g, 5.43 mmol) was dissolved in 1,4-dioxane (44 mL) and 1 M aqueous NaOH (30 mL) was added, and reaction mixture was heated at 55 °C for 18 h. The reaction mixture was cooled then neutralized with 1N HCl. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (methanol/dichloromethane, 5%) to yield 18 (1.9 g, 90%). 1H NMR (300 MHz, DMSO-d 6): δ = 10.65 (s, 1 H), 7.83 (s, 1 H), 7.37 (d, J = 1.32 Hz, 5 H), 6.61 (br s, 2 H), 5.72 (s, 1 H), 5.00–5.33 (d, J = 45 Hz, 1 H), 4.56–4.76 (m, 1 H), 4.05–4.46 (m, 3 H), 3.75–4.03 (m, 3 H). 13C NMR (75.47 MHz, DMSO-d 6): δ = 156.8, 153.9, 151.3, 137.3, 135.2, 128.9, 128.0, 126.1, 116.1, 101.1, 86.4, 84.4, 74.0, 67.8, 67.0, 64.7, 52.6, 52.2. 19F NMR (282 MHz, DMSO-d 6): δ = –200.39 (m, H-F coupled). HRMS (ESI): m/z [M + H]+ calcd for C18H19FN5O4: 388.1421; found: 388.1429.
- 19 1,5-Anhydro-2,3-dideoxy-6-O-(4,4′-dimethoxytrityl)-3-fluoro-2-(N-2-isobutyrylguanin-9-yl)-d-altritol (21). Compound 20 (1.4 g 3.79 mmol) was dissolved in anhydrous pyridine (32 mL), 4,4′-dimethoxytrityl chloride (1.7 g, 5.02 mmol) was added and the mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated in vacuo and the residue was redissolved in ethyl acetate (50 mL). The organic solution was washed with saturated aqueous NaHCO3 (50 mL) and brine (50 mL), dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. Purification by silica gel column chromatography (methanol/dichloromethane, 0–5%) yielded 21 (1.6 g, 88%). 1H NMR (300 MHz, DMSO-d 6): δ = 12.12 (s, 1 H), 11.74 (s, 1 H), 8.04–8.34 (m, 1 H), 7.36–7.50 (m, 2 H), 7.16–7.35 (m, 7 H), 6.89 (dd, J = 2.74, 8.93 Hz, 4 H), 5.75 (s, 1 H), 5.18–5.32 (m, 1 H), 4.98–5.18 (m, 1 H), 4.96 (br d, J = 3.59 Hz, 1 H), 4.77 (br d, J = 1.97 Hz, 1 H), 4.32 (br d, J = 12.93 Hz, 1 H), 4.02–4.23 (m, 1 H), 3.65–3.88 (m, 8 H), 3.47–3.65 (m, 1 H), 2.64–2.90 (m, 1 H), 1.13 (d, J = 6.73 Hz, 6 H). 13C NMR (75 MHz, CDCl3): δ = 180.4, 158.5, 158.5, 158.2, 155.8, 149.6, 148.8, 144.7, 136.2, 130.1, 128.3, 128.1, 127.8, 127.7, 127.5, 126.5, 119.5, 118.1, 113.1, 112.8, 85.9, 62.9, 62.8, 57.7, 55.2, 54.9, 36.1, 21.3, 19.1, 18.9, 18.7. 19F NMR (282 MHz, CDCl3): δ = –203.96. HRMS (ESI): m/z [M + H]+ calcd for C36H39FN5O7: 672.2834; found: 672.2836.