Site-specific incorporation of 5′-methyl DNA enhances the therapeutic profile of gapmer ASOs

Abstract We recently showed that site-specific incorporation of 2′-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5′-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5′-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2′-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5′-Me and R-5′-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5′-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.


1) Synthesis of Monomers
General. All reagents were purchased from commercial vendors and used without any further purification. Unless specified otherwise, glassware was dried in an oven and the reactions were carried out under an atmosphere of nitrogen. 1 H-NMR spectra were obtained on a Bruker 300 MHz instrument. Low resolution mass spectrometry analysis were carried out on an Agilent 1100 series LCMS system equipped with a S.E.D.E.R.E. (France) Sedex 75 Evaporative Light Scattering detector.
After cooling to 0°C, ethyl acetate was added, followed by water. The aqueous layer was removed, and the organic layer was washed with saturated sodium bicarbonate solution, brine, dried over Na2SO3 filter and concentrated under reduced pressure to a crude yellow oil.
The aqueous layer was extracted with ethyl acetate (2x) and the combined organics were washed with saturate NaHCO3 solution, brine, dried over Na2SO4, filtered and concentrated under reduced pressure to a crude oil. Purification by flash chromatography (silica gel, 220g col, 0-2% dichloromethane/methanol) afforded the desired product as a white solid. 7.8 g, 100 % yield. Compound 55. 4-Nitrobenzoate 54 (6.90 g, 11.20 mmol) was dissolved in methanol (100 mL), followed by addition of K2CO3 (3.88 g, 28.1mmol, 2.5). The reaction was stirred for 16 hours at room temperature. The solvent was removed under reduced pressure to obtained a white solid.
The solid was suspended in water (50 ml), filtered and rinsed with diethyl ether (50 mL) to atmosphere of nitrogen and cooled with an ice bath. Trimethylsilyl chloride (7.50 mL, 59 mmol, 7 eq.) was added dropwise. The ice bath was removed and the reaction was stirred for 1 hr at room temperature. The reaction again cooled with an ice bath, followed by dropwise addition of isobutyryl chloride (5.44 mL, 55.7 mmol, 7 eq.) and stirred for a further16 hours letting reaction warm up slowly to room temperature. The reaction was cooled with an ice bath, and water (30 mL) was added dropwise while keeping temperature below 5 o C. Stirring was continued at room temperature for 1 hour. The reaction was cooled in an ice bath, followed by dropwise addition of aqueous NH4OH (20 mL) and stirred for another 30 minutes. Evaporation of most of the aqueous NH4OH at room temperature was achieved under reduce pressure. The remaining solution was diluted with EtOAc and washed with water 100 (ml). The aqueous layer was discarded, and the organics were washed with saturated NaHCO3, brine, dried over Na2SO4, filtered and concentrated under reduced pressure. Purification by flash chromatography (silica gel, 100g col, 0-5% Methanol/Dichloromethane) afforded the desired product as a white solid. Compound 57. Alcohol 56 (1.20 g, 2.58 mmol) was co-evaporated with toluene (2x40 mL) under reduced pressure at 60°C. This was then dissolved into dry pyridine (50 mL) and 2,6lutidine (1.50 mL, 10.30 mmol, 4 eq.) was added. DMT-Cl (3.49 g, 10.3 mmol 4 eq.) and silver nitrate (1.7 g, 10 mmol 3.8 eq.) were then added and the reaction was heated to 45°C for 16 hours. The reaction was cooled in an ice bath and ethyl acetate (50 mL) was added, followed by water (50 mL) and stirred for 10 minutes. The aqueous layer was removed, and the organics were  63, 146.47, 136.79, 130.28, 129.14, 128.21, 127.78, 126.92, 113.18, 113.08, 113.05, 91.74, 86.12, 84.51, 72.78, 69.88, 55.27, 55.26, 40.82, 36.33, 25.76, 18.69, 18.41, 17.88, 17 Compound 58. Triethylamine (0.69 mL, 4.98 mmol, 2.5 eq) was added to a solution of compound 57 (1.53 g, 1.99 mmol) in dry THF (20 mL). The reaction was cooled in an ice bath.
The residue was resuspended in DCM (50 ml) and was transferred to a separatory funnel. The reaction mixture was washed with saturated aqueous NaHCO3, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (flash chromatography) using a DCM/EtOAc gradient. Yield: 2.3 grams (98 % 10.3 mmol, 3 eq.), and the reaction was stirred vigorously overnight at room temperature. The solvent was removed under reduced pressure. The residue was resuspended in EtOAc (50 ml) and was transferred to a separatory funnel. The reaction was washed with water, followed by brine (50 ml each), dried over MgSO4, and concentrated under reduced pressure.