Antibiotics, Vol. 12, Pages 25: Synthesis, Antibacterial and Antiribosomal Activity of the 3C-Aminoalkyl Modification in the Ribofuranosyl Ring of Apralogs (5-O-Ribofuranosyl Apramycins)

4.1. General Experimental

All reagents and solvents were purchased from commercial suppliers and were used without further purification unless otherwise specified. All experiments were carried out under a dry argon atmosphere unless otherwise specified. Unless noted otherwise, progress of reactions was monitored by thin-layer chromatography on pre-coated aluminum-backed silica gel plates (Merck Kieselgel 60F254, Merck, Darmstadt, Germany) and were visualized by UV light (254 nm) and by charring with sulfuric acid in ethanol (20:80, v/v), or potassium permanganate solution [preparation: 1.5 g of KMnO4, 10 g of K2CO3, 1.25 mL of 10% sol. of NaOH in 200 mL of H2O], or vanillin solution [preparation: 15 g of vanillin in 250 mL of ethanol and 2.5 mL of conc. H2SO4]. Flash column chromatography was performed using an IsoleraTM automated flash purification system (Biotage AB, Uppsala, Sweden) equipped with KP-Sil 10–100 g flash cartridges (Biotage AB, Uppsala, Sweden) for normal phase separations and C18 25 μm flash cartridges (Biotage AB, Uppsala, Sweden) for reverse phase separations. Optical rotations were measured at 589 nm and 20 °C on a digital polarimeter with a path length of 10 cm. 1H and 13C NMR spectra of all compounds were recorded using at 400 MHz and 600 MHz instruments unless otherwise specified and assignments made with the help of COSY, HMBC, and HSQC spectra. ESI-HRMS were recorded using a time-of-flight mass spectrometer fitted with an electrospray source. Copies of 1H and 13C NMR spectra for all new compounds are provided in the Supplementary Material.

4.2. 5-Azido-5-deoxy-1,2-O-isopropylidene-α-D-xylofuranose (8)The title compound was prepared according to literature procedure [49]. Accordingly, a stirred solution of 1,2-O-isopropylidine-α-D-xylofuranose 7 (5 g, 26.29 mmol, 1 equiv) in anhydrous dichloromethane (100 mL) was cooled to 0 °C (crushed ice bath) and treated with anhydrous pyridine (4.89 mL, 60.46 mmol, 2.3 equiv) under argon atmosphere. Then, a solution of SOCl2 (2.19 mL, 30.23 mmol, 1.15 equiv) in anhydrous dichloromethane (20 mL) was added dropwise at 0 °C over a period of 20 min. The resulting yellowish solution was stirred at 0 °C for 2 h, and the reaction progress was monitored by GC-MS assay. Upon completion of the reaction, a solution was transferred to a separatory funnel and washed with water (3 × 50 mL). The DCM layer was dried over Na2SO4, filtered off concentrated under reduced pressure keeping the water bath temperature below 30 °C to avoid product decomposition. The yellow residue was dissolved in anhydrous DMF (50 mL) and NaN3 (5.12 g, 78.9 mmol, 3 equiv) was added. The resulting brown suspension was heated at 110 °C with stirring for 18 h, then it was cooled to ambient temperature and all volatiles were removed in vacuo. The residue was dissolved in EtOAc (100 mL) and washed with water (100 mL). The water layer was back-extracted with Et2O (3 × 100 mL). The combined EtOAc and Et2O extracts were washed with water (100 mL) to remove residual DMF and inorganic salts, then dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting yellow oily residue was purified on Biotage SNAP KP-Sil 50 g silica cartridge (gradient elution from 100% petroleum ether (PE) to 45% EtOAc/PE) to give 8 (4.85 g, 86%) as a colorless sticky mass. 1H NMR (400 MHz, CDCl3, ppm) δ 5.95 (d, J = 3.7 Hz, 1H), 4.52 (d, J = 3.7 Hz, 1H), 4.31–4.23 (m, 2H), 3.66–3.57 (m, 2H), 2.22 (d, J = 5.2 Hz, 1H), 1.50 (s, 3H), 1.32 (d, J = 0.8 Hz, 3H). The 1H NMR spectrum was in agreement with that reported in the literature [50]. 4.3. 5-Azido-5-deoxy-1,2-O-isopropylidene-α-D-erythro-pentofuranos-3-ulose (9)A stirred colorless solution of xylofuranose 8 (0.80 g, 3.72 mmol) in anhydrous dichloromethane (10 mL) was cooled to 0 °C (crushed ice bath), and Dess-Martin periodinane (2.05 g, 4.83 mmol, 1.3 equiv) was added under argon atmosphere. After stirring at 0 °C for 20 min, the white suspension was warmed to ambient temperature and stirred for additional 2 h. The reaction progress was monitored by GC-MS assay. Upon completion of the reaction, the yellowish suspension was diluted with 10% aqueous sodium thiosulfate solution (30 mL) and transferred to a separation funnel. Layers were separated and the organic layer was washed with saturated aqueous NaHCO3 solution (50 mL), brine, dried over Na2SO4 and filtered. Concentration under reduced pressure afforded yellowish residue that was purified on Biotage SNAP KP-Sil 25 g silica cartridge (gradient elution from 100% PE to 40% EtOAc/PE) to give 9 (0.76 g, 96%) as a colorless oil. 1H NMR (400 MHz, CDCl3, ppm) δ 6.15 (d, J = 4.4 Hz 1H), 4.50 (td, J = 3.3, 1.1 Hz, 1H), 4.38 (dd, J = 4.4, 1.1 Hz, 1H), 3.68 (dd, J = 13.2, 3.3 Hz, 1H), 3.54 (dd, J = 13.2, 3.3 Hz, 1H), 1.49 (s, 3H), 1.43 (s, 3H). The 1H NMR spectrum was in agreement with that reported in the literature [51]. 4.4. 2-(5-Bromopentyloxy)-tetrahydro-2H-pyran (10)To a stirred solution of 5-bromo-1-pentanol (7.0 mL, 57.83 mmol) in anhydrous DCM (75 mL) was added p-toluenesulfonic acid hydrate (1.10 g, 5.78 mmol, 0.1 equiv) under argon atmosphere. The resulting clear solution was cooled to 0 °C (crushed ice bath) and 3,4-dihydro-2H-pyran (7.9 mL, 86.74 mmol, 1.5 equiv) was added dropwise over a period of 20 min. The resulting colorless solution was warmed to ambient temperature and stirred for 18 h. The reaction progress was monitored by GC-MS assay. After complete conversion, the reaction mixture was diluted with water (100 mL), layers were separated and the aqueous layer was back-extracted with DCM (3 × 50 mL). The combined DCM extracts were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The yellowish oily residue was purified on Biotage SNAP KP-Sil 100 g silica cartridge (gradient elution from 100% PE to 5% EtOAc/PE) to give 10 (12.74 g, 88%) as a colorless oil. 1H NMR (400 MHz, CDCl3, ppm) δ 4.64–4.52 (m, 1H), 3.91–3.83 (m, 1H), 3.79–3.71 (m, 1H), 3.53–3.47 (m, 1H), 3.45–3.36 (m, 3H), 1.93–1.79 (m, 3H), 1.76–1.47 (m, 9H). The 1H NMR spectrum was in agreement with that reported in the literature [28]. 4.5. (5-((Tetrahydro-2H-pyran-2-yl)oxy)pentyl)magnesium bromide (11)An oven-dried round-bottom two neck flask equipped with magnetic stir-bar was cooled to ambient temperature under an argon atmosphere. Magnesium turnings (2.66 g, 101.13 mmol, 2 equiv) were placed in the flask and activated by intensive stirring for 12 h under argon atmosphere at ambient temperature. Anhydrous THF (5 mL) was then added, a reflux condenser was mounted and the slurry was heated at 60 °C (water bath) under an argon atmosphere. 1,2-Dibromomethane (435 µL, 0.1 equiv) was added dropwise under argon, and after gas evolution ceased, a solution of bromide 10 (12.74 g, 50.57 mmol, 1 equiv) in anhydrous THF (50 mL) was added dropwise at 60 °C over a period of 45 min. The resulting gray suspension was stirred at ambient temperature for additional 3 h, then stirring was turned off and the suspension was left undisturbed overnight under argon atmosphere. The supernatant was carefully transferred via cannula to an oven-dried round-bottom flask and diluted with anhydrous THF (45 mL). Concentration of the Grignard reagent 11 was determined to be 0.38 M by titration with menthol and 1,10-phenanthroline [52]. 4.6. 5-Azido-5-deoxy-1,2-O-isopropylidene-3-C-(6-(5-((tetrahydro-2H-pyran-2-yl)oxy)pentyl)-α-D-ribofuranose (12)

Grignard reagent 11 (0.38 M solution in THF, 34.0 mL, 13.6 mmol, 2 equiv), ZnCl2 (0.7 M solution in anhydrous THF, 3.9 mL, 2.7 mmol, 0.4 equiv) and LiCl (0.5 M solution in anhydrous THF, 27.2 mL, 13.6 mmol, 2 equiv) were mixed and the resulting gray solution was stirred at ambient temperature for 30 min, whereupon it was cooled to −78 °C (dry ice/acetone bath). A solution of ketone 9 (1.45 g, 6.80 mmol) in anhydrous THF (2.5 mL) was added rapidly at a rate to keep temperature below −60 °C. The resulting yellow suspension was stirred at −78 °C for 1 h, warmed to ambient temperature over a period of 30 min and quenched with saturated aqueous NH4Cl solution (25 mL). The yellow slurry was transferred to a separation funnel, diluted with water (100 mL), and the product was back-extracted with EtOAc (3 × 50 mL). The organic extracts were combined, dried over Na2SO4 and filtered off. The solvent was evaporated under reduced pressure and the yellow residue was purified on a KP-Sil 50 g silica cartridge (gradient elution from 10% EtOAc/PE to 50% EtOAc/PE) to give 12 (1.88 g, 72%) as a yellowish viscous oil; analytical TLC on silica gel, 1:1 EtOAc/PE, Rf = 0.60. [α]D20 +21.5 (c 0.40, CHCl3). 1H NMR (400 MHz, CDCl3, ppm) δ 5.78 (d, J = 3.9 Hz, 1H), 4.59–4.55 (m, 1H), 4.31 (d, J = 3.9 Hz, 1H), 3.90 (dd, J = 7.0, 4.8 Hz, 1H), 3.85 (dt, J = 7.5, 3.6 Hz, 1H), 3.74 (dtd, J = 9.2, 7.0, 2.4 Hz, 1H), 3.53–3.46 (m, 1H), 3.45–3.35 (m, 3H), 2.61 (s, 1H), 1.88–1.78 (m, 1H), 1.76–1.69 (m, 1H), 1.64–1.48 (m, 8H), 1.58 (s, 3H), 1.45–1.36 (m, 4H), 1.37 (s, 3H). 13C NMR (101 MHz, CDCl3, ppm) δ 112.7, 103.8, 99.0, 81.7, 80.5, 79.0, 67.6, 62.5, 49.7, 30.9, 30.7, 29.8, 27.0, 26.7, 26.6, 25.6, 22.9, 19.8. HRMS (ESI/Q-TOF) m/z: [M-acetone + H]+ Calculated C15H26N3O5: 328.3848. Found: 328.3822.

4.7. 5-Azido-5-deoxy-1,2-O-isopropylidene-3-C-(6-(5-((tetrahydro-2H-pyran-2-yl)oxy)pentyl)-3-O-benzoyl-α-D-ribofuranose (13)

Benzoic anhydride (2.13 g, 9.42 mmol, 3 equiv) and DMAP (192 mg, 1.57 mmol, 0.5 equiv) were added to a stirred solution of tertiary alcohol 12 (1.21 g, 3.12 mmol) in anhydrous pyridine (15 mL) at 0 °C (crushed ice bath). The resulting yellowish solution was heated at 100 °C for 18 h. After cooling to ambient temperature, volatiles were evaporated under reduced pressure. The yellow residue was diluted with EtOAc (50 mL) and washed with saturated aqueous NaHCO3 solution (3 × 50 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The yellow oily residue was purified on KP-Sil 50 g silica cartridge (gradient elution from 10% EtOAc/PE to 50% EtOAc/PE) to give 13 (1.43 g, 93%) as a yellowish sticky mass; analytical TLC on silica gel, 1:1 EtOAc/PE, Rf = 0.63. [α]D20 +22.8 (c 0.57, CHCl3).1H NMR (400 MHz, CDCl3, ppm) δ 8.04–7.98 (m, 2H), 7.62–7.55 (m, 1H), 7.49–7.42 (m, 2H), 5.81 (d, J = 3.7 Hz, 1H), 4.94 (d, J = 3.7 Hz, 1H), 4.55–4.49 (m, 1H), 4.37 (dd, J = 7.2, 4.8 Hz, 1H), 3.82 (ddd, J = 11.1, 7.6, 3.3 Hz, 1H), 3.74–3.65 (m, 1H), 3.63–3.57 (m, 2H), 3.47 (dt, J = 10.4, 5.0 Hz, 1H), 3.34 (dt, J = 9.5, 6.3 Hz, 1H), 2.03–1.87 (m, 2H), 1.83–1.64 (m, 2H), 1.60–1.46 (m, 9H), 1.45–1.29 (m, 7H).13C NMR (101 MHz, CDCl3, ppm) δ 165.0, 133.4, 130.1, 129.9, 128.6, 112.9, 104.0, 99.0, 85.3, 83.1, 80.0, 67.4, 62.5, 50.2, 30.9, 30.4, 29.5, 27.0, 26.9, 26.8, 25.6, 23.6, 19.8. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calculated C25H35N3O7Na: 512.2373. Found: 512.2394.

4.8. 5-Azido-5-deoxy-1,2-O-isopropylidene-3-C-(5-hydroxypentyl)-3-O-benzoyl-α-D-ribofuranose (14)

Tetrabutylammonium tribromide (80 mg, 0.17 mmol, 0.1 equiv) was added to a stirred solution of THP-protected alcohol 13 (810 mg, 1.65 mmol) in MeOH (15 mL) at ambient temperature. The resulting orange solution was stirred for 3 h, then acetone (25 mL) was added and the resulting solution was stirred for additional 15 min. After the volatiles were evaporated under reduced pressure, the orange oily residue was diluted with EtOAc (50 mL) and washed with saturated aqueous NaHCO3 solution (3 × 50 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The orange oily residue was purified on KP-Sil 50 g silica cartridge (gradient elution from 20% EtOAc/PE to 60% EtOAc/PE) to give 14 (655 mg, 98%) as a yellowish viscous oil; analytical TLC on silica gel, 1:1 EtOAc/PE, Rf = 0.30. [α]D20 +70.2 (c 0.38, CHCl3). 1H NMR (400 MHz, CDCl3, ppm) δ 8.03–7.98 (m, 2H), 7.62–7.55 (m, 1H), 7.49–7.42 (m, 2H), 5.81 (d, J = 3.7 Hz, 1H), 4.94 (d, J = 3.7 Hz, 1H), 4.37 (dd, J = 7.3, 4.8 Hz, 1H), 3.63–3.56 (m, 4H), 2.03–1.88 (m, 2H), 1.58–1.51 (m, 2H), 1.49 (s, 3H), 1.43–1.29 (m, 8H). 13C NMR (101 MHz, CDCl3, ppm) δ 165.0, 133.4, 130.0, 129.9, 128.6, 112.9, 104.0, 85.2, 83.1, 79.9, 62.8, 50.1, 32.4, 30.4, 26.8, 26.7, 26.3, 23.6. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calculated C20H27N3O6Na: 428.1798. Found: 428.1786.

4.9. 5-Azido-5-deoxy-1,2-O-isopropylidene-3-C-(5-oxopentyl)-3-O-benzoyl-α-D-ribofuranose (15)

A stirred solution of alcohol 14 (610 mg, 1.50 mmol) in anhydrous DCM (10 mL) was cooled to 0 °C (crushed ice bath) under argon atmosphere and treated with Dess-Martin periodinane (830 mg, 1.96 mmol, 1.3 equiv), followed by few drops of NEt3. After stirring at 0 °C for 20 min, the white suspension was warmed to ca. 10 °C and stirred at this temperature for additional 2–3 h. The progress of the reaction was followed by TLC and UPLC assays. After completion of the reaction, the white suspension was diluted with 10% aqueous sodium thiosulfate solution (25 mL) and layers were separated. The organic layer was washed with saturated NaHCO3 solution (50 mL), brine, dried over Na2SO4 and filtered. Removal of volatiles under reduced pressure afforded pale yellow oily residue that was purified on KP-Sil 10 g silica cartridge (gradient elution from 20% to 50% EtOAc/PE) to afford 15 (572 mg, 94%) as a colorless oil; analytical TLC on silica gel, 1:1 EtOAc/PE, Rf = 0.51. [α]D20 +69.1 (c 0.42, CHCl3). 1H NMR (400 MHz, CDCl3, ppm) δ 9.73 (t, J = 1.4 Hz, 1H), 8.03–7.98 (m, 2H), 7.63–7.56 (m, 1H), 7.48–7.44 (m, 2H), 5.81 (d, J = 3.7 Hz, 1H), 4.93 (d, J = 3.7 Hz, 1H), 4.36 (dd, J = 6.9, 5.1 Hz, 1H), 3.63–3.55 (m, 2H), 2.46–2.40 (m, 2H), 2.06–1.93 (m, 2H), 1.67–1.58 (m, 2H), 1.49 (s, 3H), 1.40–1.32 (m, 5H). 13C NMR (101 MHz, CDCl3, ppm) δ 201.8, 165.0, 133.5, 129.9, 129.9, 128.6, 113.0, 103.9, 85.1, 83.1, 79.8, 50.0, 43.5, 30.3, 26.8, 23.3, 22.4. HRMS (ESI/Q-TOF) m/z: [M-acetone + H]+ Calculated C17H20N3O5: 346.1403. Found: 346.1414.

4.10. 5-Azido-5-deoxy-1,2-O-isopropylidene-3-C-(5-(dimethylamino)pentyl)-3-O-benzoyl-α-D-ribofuranose (16)

To a solution of aldehyde 15 (570 mg, 1.41 mmol) in anhydrous THF (15 mL) at ambient temperature were added Me2NH (2 M solution in THF, 2.1 mL, 4.2 mmol, 3 equiv) and glacial acetic acid (81 µL, 1.41 mmol, 1 equiv). The resulting yellow solution was stirred for 1 h then cooled to 0 °C (crushed ice bath), and NaBH(OAc)3 (449 mg, 2.12 mmol, 1.5 equiv) was added in 3 portions. The yellow suspension was stirred at 0 °C for 2 h whereupon water (25 mL) and saturated aqueous NaHCO3 solution (25mL) were added. The resulting cloudy solution was extracted with EtOAc (3 × 30 mL), combined organic extracts were dried over Na2SO4, filtered and concentrated under reduced pressure. The yellow oily residue was purified on KP-Sil 10 g silica cartridge (gradient elution from 100% EtOAc to 2% NEt3 in EtOAc) to give 16 (440 mg, 72%) as a yellow oil; analytical TLC on silica gel, 30% MeOH in DCM, Rf = 0.33. [α]D20 +48.0 (c 0.39, CHCl3). 1H NMR (400 MHz, CDCl3, ppm) δ 8.03–7.99 (m, 2H), 7.61–7.56 (m, 1H), 7.49–7.43 (m, 2H), 5.81 (d, J = 3.7 Hz, 1H), 4.94 (d, J = 3.7 Hz, 1H), 4.36 (dd, J = 7.4, 4.8 Hz, 1H), 3.64–3.55 (m, 2H), 2.21–2.16 (m, 8H), 2.05–1.88 (m, 2H), 1.49 (s, 3H), 1.45–1.38 (m, 2H), 1.37–1.25 (m, 7H). 13C NMR (101 MHz, CDCl3, ppm) δ 165.0, 133.4, 130.1, 129.9, 128.6, 112.9, 104.0, 85.3, 83.1, 80.0, 59.7, 50.2, 45.6, 30.4, 28.0, 27.5, 26.8, 26.8, 26.7, 23.7. HRMS (ESI/Q-TOF) m/z: [M + H]+ Calculated C22H33N4O5: 433.2451. Found: 433.2462.

4.11. 1,2-Di-O-acetyl-5-azido-5-deoxy-3-C-(5-(dimethylamino)pentyl)-3-O-benzoyl-α/β-D-ribofuranose (17)

A stirred solution of 1,2-O-isopropylidene-protected ribofuranose 16 (1.1 g, 2.54 mmol) in glacial acetic acid (20 mL) and Ac2O (9.6 mL, 102 mmol, 40 equiv) was cooled to 0 °C (crushed ice bath). Concentrated H2SO4 (68 µL, 1.27 mmol, 0.5 equiv) was added dropwise at 0 °C. The yellow solution was warmed to ambient temperature and stirred for 18 h, whereupon volatiles were removed under reduced pressure keeping the water bath temperature below 30 °C. The brown oily residue was diluted with DCM (50 mL) and water (20 mL), cooled to 0 °C (crushed ice bath) and pH of aqueous layer was adjusted to the neutral by addition of saturated aqueous NaHCO3 solution. The mixture was transferred to a separation funnel, layers were separated and the aqueous layer was extracted with EtOAc (3 × 30 mL). The combined organic extracts were dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting brown oily residue was purified on KP-Sil 25 g silica cartridge (gradient elution from 100% EtOAc to 2% NEt3 in EtOAc) to give 17 (1.01 g, 83%; 3:2 α:β mixture of anomers) as a yellow oil; analytical TLC on silica gel, 5% NEt3 in EtOAc, Rf = 0.40. 1H NMR (400 MHz, CDCl3, ppm) δ 8.09–7.94 (m, 2H, both anomers), 7.64–7.57 (m, 1H, both anomers), 7.51–7.43 (m, 2H, both anomers), 6.49 (d, J = 4.6 Hz, 0.6H, major anomer), 6.23 (d, J = 2.4 Hz, 0.4H, minor anomer), 5.61 (d, J = 2.4 Hz, 0.4H, minor anomer), 5.32 (d, J = 4.6 Hz, 0.6H, major anomer), 4.85 (dd, J = 4.8, 3.5 Hz, 0.6H, major anomer), 4.74 (dd, J = 6.6, 3.3 Hz, 0.4H, minor anomer), 3.84–3.81 (m, 0.4H, minor anomer), 3.81–3.77 (m, 0.6H, major anomer), 3.60–3.56 (m, 0.6H, major anomer), 3.56–3.53 (m, 0.4H, minor anomer), 2.63–2.45 (m, 1H, both anomers), 2.42–2.30 (m, 1H, both anomers), 2.18 (s, 6H, both anomers), 2.15 (s, 1.8H, major anomer), 2.14 (s, 1.2H, minor anomer), 2.13 (s, 1.2H, minor anomer), 2.04 (s, 1.8H, major anomer), 1.91–1.73 (m, 1H, both anomers), 1.47–1.34 (m, 3H, both anomers), 1.32–1.23 (m, 4H, both anomers). 13C NMR (101 MHz, CDCl3, ppm) δ 169.0, 165.9, 165.7, 165.4, 164.8, 164.5, 157.0, 133.6, 133.5, 133.4, 133.3, 129.9, 129.8, 129.6, 129.5, 129.3, 129.1, 129.1, 128.8, 128.6, 128.6, 128.5, 128.4, 128.3, 105.8, 100.7, 96.2, 95.6, 83.9, 82.7, 80.5, 78.5, 75.9, 75.2, 71.8, 71.0, 70.2, 69.7, 66.4, 65.8, 63.1, 60.7, 60.1, 59.1, 58.2, 57.4, 55.4, 51.2, 42.7, 42.5, 31.2, 30.2, 29.5, 27.9, 26.2, 23.6, 22.3, 19.9. HRMS (ESI/Q-TOF) m/z: [M + H]+ Calculated C71H76N17O21: 1502.5402. Found: 1502.5424.

19β-anomer:[α]D20 +61.3 (c 0.31, CHCl3).1H NMR (400 MHz, CDCl3, ppm) δ 11.2–10.9 (br s, 1H), 8.10–8.06 (m, 2H), 8.05–8.00 (m, 2H), 7.98–7.91 (m, 4H), 7.71–7.65 (m, 2H), 7.65–7.59 (m, 1H), 7.56–7.50 (m, 4H), 7.47–7.40 (m, 3H), 7.40–7.30 (m, 5H), 7.25–7.21 (m, 2H), 6.00 (t, J = 10.1 Hz, 1H), 5.75–5.67 (m, 2H), 5.43 (s, 1H), 5.30 (s, 1H), 5.24 (t, J = 9.9 Hz, 1H), 5.16 (dd, J = 10.4, 3.6 Hz, 1H), 4.80 (d, J = 5.6 Hz, 1H), 4.76 (dd, J = 6.9, 3.6 Hz, 1H), 4.68 (dd, J = 12.2, 2.4 Hz, 1H), 4.62 (dd, J = 12.2, 4.9 Hz, 1H), 4.22 (dd, J = 8.6, 2.2 Hz, 1H), 4.15–4.06 (m, 3H), 3.89 (t, J = 10.1 Hz, 1H), 3.84–3.79 (m, 1H), 3.78–3.73 (m, 2H), 3.57–3.49 (m, 3H), 3.45 (ddd, J = 12.4, 10.0, 4.8 Hz, 1H), 3.07–2.99 (m, 3H), 2.93 (s, 3H), 2.91–2.82 (m, 6H), 2.45 (dt, J = 13.0, 4.5 Hz, 1H), 2.10–1.99 (m, 1H), 1.79–1.64 (m, 7H), 1.64–1.53 (m, 2H), 1.38–1.24 (m, 4H).13C NMR (101 MHz, CDCl3, ppm) δ 169.1, 166.3, 166.1, 165.8, 165.3, 164.5, 157.4, 133.9, 133.8, 133.8, 133.7, 133.5, 130.1, 130.0, 129.9, 129.9, 129.7, 129.5, 129.0, 128.9, 128.9, 128.8, 128.7, 128.7, 128.6, 128.5, 128.4, 106.9, 100.5, 96.4, 95.9, 83.4, 83.3, 80.9, 79.3, 76.7, 75.3, 72.0, 71.4, 70.6, 70.0, 66.6, 66.0, 63.5, 61.1, 60.4, 59.3, 58.5, 58.1, 56.0, 51.3, 43.3, 43.2, 31.5, 30.4, 29.5, 28.5, 26.5, 24.1, 22.8, 20.9. HRMS (ESI/Q-TOF) m/z: [M + H]+ Calculated C71H76N17O21: 1502.5402. Found: 1502.5435.

4.13. 5-O-[5′′′-Amino-5′′′-deoxy-3-C-(5-(dimethylamino)pentyl)-β-D-ribofuranosyl]-apramycin heptaacetate (5)

A mixture of apramycin derivative 19β (80 mg, 0.05 mmol) in dioxane (1 mL) and NaOH (2 M aqueous solution, 240 µL, 0.48 mmol, 9 equiv) was heated at 80 °C for 24 h. The reaction progress was monitored by UPLC-MS assay. Upon complete conversion, the colorless solution was cooled to 0 °C (crushed ice bath) and dry ice was added portion-wise until pH 8. The resulting solution was concentrated to dryness under reduced pressure. The white residue was dissolved in a mixture of dioxane/deionized water/glacial acetic acid (1:1:1, 3 mL) and 10% Pd on carbon (85 mg, 0.08 mmol, 1.5 equiv) was added. The black suspension was vigorously stirred under 3 atm hydrogen pressure at ambient temperature for 18 h, and the progress of the reaction was monitored by UPLC-MS assay. Upon complete conversion, the black suspension was filtered through the pad of Celite®®®, and the filter cake was washed with 1:1 AcOH:water mixture. Combined filtrates were evaporated under reduced pressure, and the sticky oil was purified by reversed-phase preparative HPLC (column: XBridge® BEH Prep OBDTM Amide, 5 μm, 30 x 100 mm, Waters Corporation Ltd, Dublin, Ireland) using gradient elution from 95:5 A:B to 10:90 A:B (eluent A: 0.1% solution of AcOH in MeCN; eluent B: 0.1% solution of AcOH in water). The product-containing fractions (identified by ESI-MS) were combined and concentrated. Glacial acetic acid was added to the sticky oily residue, and subsequent trituration with MeCN afforded the heptaacetate salt of 5 as a white amorphous solid (32 mg, 50% yield). [α]D20 +64.4 (c 0.104, H2O). 1H NMR (600 MHz, D2O, ppm) δ 5.72 (d, J = 3.9 Hz, 1H), 5.41 (d, J = 3.9 Hz, 1H), 5.35 (d, J = 4.3 Hz, 1H), 5.12 (d, J = 8.5 Hz, 1H), 4.49 (t, J = 2.8 Hz, 1H), 4.09–4.04 (m, 2H), 4.02 (d, J = 4.1 Hz, 1H), 3.90 (t, J = 9.3 Hz, 1H), 3.88–3.83 (m, 2H), 3.83 (t, J = 10.1 Hz, 1H), 3.79–3.72 (m, 2H), 3.69 (dd, J = 12.4, 4.5 Hz, 1H), 3.65–3.55 (m, 3H), 3.38 (ddd, J = 13.7, 9.9, 4.1 Hz, 1H), 3.28 (dd, J = 8.5, 2.8 Hz, 1H), 3.24 (td, J = 12.5, 4.2 Hz, 1H), 3.21–3.12 (m, 2H), 3.05–2.98 (m, 3H), 2.75 (s, 6H), 2.68 (s, 3H), 2.33 (dt, J = 12.5, 4.2 Hz, 1H), 2.27 (dt, J = 9.0, 4.6 Hz, 1H), 2.01–1.92 (m, 1H), 1.82 (s, 21H), 1.73 (q, J = 12.7 Hz, 1H), 1.66–1.54 (m, 3H), 1.49–1.35 (m, 2H), 1.33–1.22 (m, 3H). 13C NMR (151 MHz, D2O, ppm) δ 180.2, 106.7, 93.8, 93.0, 92.1, 82.7, 81.6, 78.4, 77.8, 73.8, 71.1, 69.6, 69.0, 68.9, 67.7, 65.3, 62.0, 59.6, 58.7, 56.9, 51.3, 49.5, 48.1, 47.0, 41.8, 39.7, 31.0, 29.3, 27.9, 26.4, 25.4, 23.2, 22.3, 21.3. HRMS (ESI/Q-TOF) m/z: [M + H]+ Calculated C33H66N7O14: 784.4668. Found: 784.4665. Anal. Calcd for C47H93N7O28: C, 44.10; H, 7.98; N, 7.66. Found: C, 44.31; H, 7.75; N, 7.74.

4.14. 5-O-[5′′′-Amino-5′′′-deoxy-3-C-(5-(dimethylamino)pentyl)-α-D-ribofuranosyl]-apramycin hexaacetate (6)

A mixture of apramycin derivative 19α (60 mg, 0.04 mmol) in dioxane (1 mL) and NaOH (2 M aqueous solution, 180 µL, 0.36 mmol, 9 equiv) was heated at 80 °C for 24 h. The reaction progress was monitored by UPLC-MS assay. Upon complete conversion, the colorless solution was cooled to 0 °C (crushed ice bath) and dry ice was added portion-wise until pH 7–8. The resulting solution was concentrated to dryness under reduced pressure. The white residue was dissolved in a mixture of dioxane/deionized water/glacial acetic acid (1:1:1, 3 mL) and 10% Pd on carbon (64 mg, 0.06 mmol, 1.5 equiv) was added at ambient temperature. The black suspension was vigorously stirred under 3 atm hydrogen pressure at ambient temperature for 18 h, and the progress of the reaction was monitored by UPLC-MS assay. Upon complete conversion, the black suspension was filtered through the pad of CeliteTM 545 (Thermo Fischer Scientific, Waltham, MA, USA), and the filter cake was washed with 1:1 AcOH:water mixture. Combined filtrates were evaporated under reduced pressure, and the sticky oil was purified by reversed-phase preparative HPLC (column: XBridge® BEH Prep OBDTM Amide, 5 μm, 30 x 100 mm, Waters Corporation Ltd, Dublin, Ireland) using gradient elution from 95:5 A:B to 10:90 A:B (eluent A—0.1% solution of AcOH in MeCN; eluent B—0.1% solution of AcOH in water). The product-containing fractions (identified by ESI-MS) were combined and concentrated. Glacial acetic acid was added to the sticky oily residue, and subsequent trituration with MeCN afforded the hexaacetate salt of 6 as a white amorphous solid (31 mg, 65% yield). [α]D20 +76.9 (c 0.25, H2O). 1H NMR (600 MHz, D2O, ppm) δ 5.60 (d, J = 3.5 Hz, 1H), 5.49 (d, J = 3.9 Hz, 1H), 5.45 (d, J = 4.6 Hz, 1H), 5.18 (d, J = 8.5 Hz, 1H), 4.53 (t, J = 2.8 Hz, 1H), 4.16 (dd, J = 11.4, 2.5 Hz, 1H), 4.07 (d, J = 4.5 Hz, 1H), 3.95–3.82 (m, 6H), 3.81–3.75 (m, 3H), 3.71 (dd, J = 9.8, 3.9 Hz, 1H), 3.50 (dt, J = 12.5, 4.2 Hz, 1H), 3.33–3.17 (m, 4H), 3.15–3.07 (m, 4H), 2.88 (s, 6H), 2.74 (s, 3H), 2.35–2.26 (m, 2H), 2.01–1.93 (m, 1H), 1.92 (s, 18H), 1.79–1.70 (m, 2H), 1.70–1.59 (m, 2H), 1.58–1.47 (m, 2H), 1.43–1.36 (m, 3H). 13C NMR (151 MHz, D2O, ppm) δ 181.3, 107.5, 95.0, 94.5, 93.2, 83.4, 82.3, 78.0, 72.4, 70.6, 70.4, 69.7, 66.5, 66.3, 63.0, 60.5, 59.8, 57.6, 52.1, 50.5, 49.0, 48.1, 42.5, 40.5, 31.8, 30.5, 30.3, 28.2, 26.0, 23.8, 23.2, 22.0. HRMS (ESI/Q-TOF) m/z: [M + H]+ Calculated C33H66N7O14: 784.4668. Found: 784.4676. Anal. Calcd for C45H89N7O26: C, 44.18; H, 8.06; N, 8.01. Found: C, 44.07; H, 7.71; N, 7.69.

4.15. Cell-Free Luciferase Translation Assays

Cell-free in vitro translation inhibition assays were performed using luciferase mRNA and bacterial S30 extracts containing either wild-type bacterial or human hybrid ribosomes. In brief, firefly luciferase mRNA was transcribed in vitro using T7 RNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) using a plasmid as template in which the mammalian promoter in pGL4.14 has been replaced by theT7 bacteriophage promoter (Promega, USA). Test articles in aqueous solution containing 0.3% Tween20 were dispensed into white 96-well plates (Eppendorf, Germany) using the TECAN D300e digital dispenser (Tecan, Switzerland). The test article dispensing volume was balanced to a total of 1.5 µL by 0.3% Tween20 in water. The reaction volume was brought to 15 µL by addition of 13.5 µL Translation Master Mix comprised of bacterial S30 extract, 0.2 mM amino acid mix, 6 µg tRNA (Sigma-Aldrich, USA), 0.4 µg hFluc mRNA, 0.3 µL protease inhibitor (cOmplete, EDTA-free, Roche, USA), 12 U RNAse inhibitor (Ribolock, Thermo Fisher Scientific, Waltham, MA, USA), and 6 µL S30 premix without amino acids (Promega, USA). Dispensing and mixing of reagents was performed on ice prior to incubating the sealed plates at 37 °C. After 1 h of incubation, the reaction was stopped on ice and 75 µL of luciferase assay reagent (Promega, USA) was added to each well. Luminescence was recorded with a plate reader (BIO-TEK FLx800, Witec AG, Littau, Switzerland).

4.16. Antibacterial Inhibition AssaysThe minimal inhibitory concentrations (MIC) of synthesized compounds were determined by broth microdilution assays according to CLSI reference methodology M07 [53] as described previously [6] and using strains described previously [54]. Clinical bacterial isolates were obtained from the diagnostic laboratories of the Institute of Medical Microbiology, University of Zurich.

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