Potent inhibition of miR-27a by neomycin–bisbenzimidazole conjugates† †Electronic supplementary information (ESI) available: Experimental details and synthesis of compounds. See DOI: 10.1039/c5sc01969a

Potent downregulation of oncogenic miRNA is obtained by conjugation of neomycin and bisbenzimidazoles.


Table of Contents
To a solution of 1a (40.0 mg, 86.3 mol) in dry THF (20.0 mL), NaH was added (50.0 mg, 2.00 mmol) at 0  C. After 15 min stirring, (Boc) 2 O (100 mg, excess) was added to the mixture and it was allowed to slowly warm to room temperature . After 5h, the reaction mixture was concentrated under the stream of nitrogen. The crude product was purified by column chromatography on silica gel (5 micron mesh size) using dichloromethane-methanol (0-20 % methanol in dichloromethane) as eluent to obtain the desired product which eluted as two rotamers which moved distinctly during the TLC analysis. The rotamer I (R f = 0.68 in dichloromethane:methanol 8.5:1.5 v/v) was obtained in 32 % yield and rotamer II (R f = 0.59 in dichloromethane:methanol 8

Synthesis of 2b.
To a solution of 2a (60.0 mg, 0.12 mmol) in dry THF (20 mL) NaH was added (97.0 mg, 3.88 mmol) at 0  C. After 30 min stirring, (Boc) 2 O (0.1 g, 0.45 mmol) was added to the solution and the mixture was allowed to slowly warm to room temperature . After overnight stirring, the reaction mixture was concentrated under the stream of nitrogen and mixed with some silica powder (5 micron mesh size).

Synthesis of 2.
To a solution of 2b (rotamer I, R f = 0.53 in dichloromethane:methanol 9:1 v/v, 26.6 mg, 37.7 mol) in ethanol (3.00 mL), a solution of Boc protected neomycin-azide (47.0 mg, 37.7 mol) in ethanol (1.00 mL) was added followed by by addition of a freshly made copper(I) catalyst made by adding aqueous solutions of CuSO 4 (1.6 mg, 10.0 mol) and sodium ascorbate (4.0 mg, 20.0 mol). The reaction mixture was stirred for 12h at room temperature. TLC on silica gel indicated formation of the product.

Synthesis of 3.
To a solution of 3b (rotamer I 28.7 mg, 37.0 mol) in ethanol (3.50 mL), a solution of Boc protected neomycin-azide (46.0 mg, 37.0 mol) in ethanol (1.00 mL) was added followed by addition of a freshly made copper(I) catalyst made by adding aqueous solutions of CuSO 4 (1.60 mg, 10.0 mol) and sodium ascorbate (3.96 mg, 20.0 mol). The reaction mixture was stirred for 42h at room temperature.
TLC on silica gel indicated formation of the product. Solvents were evaporated under reduced pressure. The crude mixture was purified by column chromatography on silica gel (5 micron, mesh size) using dichloromethane-ethanol (0-12 % ethanol in dichloromethane) as eluent. This yielded Boc protected 3 (35.2 mg, R f = 0.45 in dichloromethane: ethanol 9:1 v/v). The starting material alkyne was also recovered (11.2 mg). The Boc protected conjugate 3 was then dissolved in dichloromethane (3.0 mL) followed by addition of trifluoroacetic acid (0.6 mL). The mixture was stirred for 2h at room temperature. Water (4.0 mL) was added and it was washed with dichloromethane (

Synthesis of 4b.
To a solution of 4a (0.11 g, 0.20 mmol) in dry THF (60.0 mL), NaH was added (0.05 g, 2.08 mmol) at 0  C. After stirring for 30 min on ice, (Boc) 2 O (0.20 g, 0.90 mmol) was added to the solution and the mixture was allowed to slowly warm to room temperature . The stirring was continued overnight following which the reaction mixture was concentrated under the stream of nitrogen. The reaction mixture was mixed with small amount of silica (5 micron mesh size). The crude product was purified by column chromatography on silica gel (5 micron mesh size) using dichloromethane-methanol (0-12 % methanol in dichloromethane) as eluent to obtain the desired product which eluted as two rotamers

Synthesis of 5.
To a solution of 5b (rotamer I, 27.9 mg, 36.5 mol) in ethanol (3.00 mL), a solution of Boc protected neomycin-azide (45.3 mg, 36.5 mol) in ethanol (1.00 mL) was added followed by addition of a freshly made copper(I) catalyst made by adding aqueous solutions of CuSO 4 (1.6 mg, 10.0 mol) and sodium ascorbate (3.9 mg, 20.0 mol). The reaction mixture was stirred for 24h at room temperature.
TLC on silica gel indicated formation of the product. Volatiles were evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (5 micron, mesh size) using dichloromethane-ethanol as eluent. This yielded Boc protected 5 (49.1 mg, R f = 0.33 in dichloromethane: methanol 9:1 v/v). This product (46.5 mg) was then dissolved in dichloromethane (2.00 mL) followed by addition of trifluoroacetic acid (0.60 mL). The mixture was stirred overnight at room temperature. Water (3.0 mL) was added to it followed which was followed by washing with dichloromethane (3 × 1 mL). The aqueous layer was lyophilized to afford the desired compound as yellow solid (32 mg

Synthesis of 6b.
To a solution of 6a (0.10 g, 0.16 mmol) in dry THF (50.0 mL), NaH was added (12.0 mg, 0.50 mmol) at 0  C. After 15 min stirring, (Boc) 2 O (0.11 g, 0.50) was added to the solution and the mixture was allowed to slowly warm to room temperature . After 5h, the reaction mixture was concentrated under the stream of nitrogen gas. Small amount of silica powder (5 micron mesh size ) was added to it. The crude product was purified by column chromatography using dichloromethane-methanol (0-15% ethanol in dichloromethane) as eluent to obtain the desired product which eluted as two different

Synthesis of 6.
To a solution of 6b (rotamer I, 27.0 mg, 34.1 mol) in ethanol (3.00 mL), a solution of Boc protected neomycin-azide (42.3 mg, 34.1 mol) in ethanol (1.00 mL) was added followed by addition of a freshly made copper(I) catalyst made by adding aqueous solutions of CuSO 4 (1.6 mg, 10.0 mol) and sodium ascorbate (3.9 mg, 20.0 mol). The reaction mixture was stirred for 24h at room temperature.
TLC on silica gel indicated formation of the product. Volatiles were evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (5 micron, mesh size) using dichloromethane-ethanol (0-12 % ethanol in dichloromethane) as eluent. This yielded Boc protected 6 (49.1 mg, R f = 0.33 in dichloromethane: methanol 9:1 v/v). This product (46.5 mg) was then dissolved in dichloromethane (2.00 mL) followed by addition of trifluoroacetic acid (0.60 mL).
The mixture was stirred overnight at room temperature. Water (3.0 mL) was added to it which was followed by washing with dichloromethane (

Synthesis of 7b.
To a solution of 7a (0.10 g, 0.16 mmol) in dry THF (40.0 mL), NaH was added (0.10 g, 4.16 mmol) at 0  C. After 15 min stirring, (Boc) 2 O (0.20 g, 0.91 mmol) was added to the solution and the mixture was allowed to slowly warm to room temperature . After 5h, the reaction mixture was concentrated under the stream of nitrogen gas and mixed with small amount of silica powder (5 micron mesh size).

Synthesis of 7.
To a solution of 7b (rotamer I, 39.2 mg, 47.9 mol) in ethanol (3.00 mL), a solution of Boc protected neomycin-azide (60.0 mg, 47.9 mol) in ethanol (1.00 mL) was added followed by addition of a freshly made copper(I) catalyst made by adding aqueous solutions of CuSO 4 (1.75 mg, 11.0 mol) and sodium ascorbate (4.35 mg, 22.0 mol) in water (0.50 mL). The reaction mixture was stirred for 24h at room temperature. TLC on silica gel indicated formation of the product. Solvents were evaporated under reduced pressure. The crude mixture was purified using column chromatography on silica gel (5 micron, mesh size) using dichloromethane-ethanol as eluent. This yielded Boc protected 7 (41.1 mg, R f = 0.36 in dichloromethane: ethanol 9:1 v/v). The product was dissolved in dichloromethane (3.00 mL) followed by addition of trifluoroacetic acid (0.60 mL). The mixture was stirred for 2h at room temperature. Water (3.00 mL) was added to it which was followed by washing it with dichloromethane (3×1 mL). The aqueous layer was lyophilized to afford the desired compound as pale yellow solid (

General scheme of synthesis for compounds 17-19.
To a solution of 15 (100 mg, 0.29 mmol) in a mixture of methanol-water (3 mL, 2:1 v/v), appropriate diazide (50 fold excess) was added followed by the addition of a freshly prepared copper catalyst {copper sulfate (7 mg, 0.043 mmol), sodium ascorbate (20 mg, 0.10 mmol)}. The mixture was stirred overnight at room temperature. Volatiles were removed under reduced pressure. Crude product was purified by column chromatography using dichloromethane-methanol (0-15% methanol in dichloromethane) as eluent to afford the desired compounds as pale yellow powder. (34.7 mmol assuming 100% alkyne in the mixture). The reaction mixture was stirred at room temperature overnight while being wrapped in aluminum foil. TLC showed formation of the product.
Volatiles were evaporated and the crude product was taken up in dichloromethane and then wet loaded on a silica gel column. Elution with dichloromethane-methanol (0-18 % methanol in dichloromethane) afforded the desired Boc protected neomycin-benzimidazole conjuagtes (8-12a) as white solid. The Boc protected conjugate was taken up in dichloromethane (3 mL), 4M-HCl in 1, 4 dioxane (0.5 mL) was added to it. After stirring at room temperature for 30 min, precipitation of the conjugate was induced by addition of small volumes (~1-2 mL) hexanes and ether. The precipitated product was centrifuged and the supernatant was discarded. The precipitated solid was taken up in water and extracted with dichloromethane (3×1 mL). The aqueous layer was lyophilized to dryness to afford the desired neomycin conjuagtes (8-12) as yellowish white solid (52-68 %) overall yield for two steps.
Note. For clarity in the 1 H NMR spectra of compounds 8-12, the residual proton signals from the deuterared solvent were suppressed.    Western blot detection of Prohibitin levels upon treatment with NH compounds 1-7 at a final concentration of 5 μM. Compound 3, 5 and 6 showed increase in PHB levels whereas; compound 1, 2 and 4 did not procure any change. AntimiR-27a (100 nM) was used as a positive control. β-Tubulin was used as an endogenous internal control.

Fig S8:
Representative histogram plots of FACS mediated cell cycle analysis upon treatment with compounds 1, 3 4 and 5 at a final concentration of 5 μM. Compound 3, 4 and 5 showed increase in G0/G1 levels and decrease in S phase levels whereas; compound 1 did not have significant change in G0/G1 phase as compared to the Hoechst 33258 control (a-e). The G2/M phase levels did not procure any significant changes after treatment with any compounds. The cells treated with Neomycin at 5 μM (g) 20 μM (h) and antimiR (i) were compared to untreated cells stained with Pi (f).