RU2394834C1 - Carbohydrate-containing cationic amphiphiles, capable of delivering nucleic acid in mammal cells - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to carbohydrate-containing polycationic amphiphiles (1-3) which are trihydrochlorides of rac-N-[6-(β-D-glycopyranosyloxy)hexyl]-N-[2,3-di(tetradecyloxy)prop-1-yl]-4-[(12-amino-4,9-diazadodec-1-yl)amino-succinylamino]benzenesulfonamide of the given general formula

, where A is a 1,2-di-O-tetradecyl-rac-glycerin residue, B is a galactose residue (for (1)), galactose (for (2)) and mannose (for (3)), C is a spermine residue, n= 6, m = 2.

EFFECT: obtaining compounds which are capable of delivering nucleic acid into mammal cells.

1 cl, 4 tbl, 2 dwg, 11 ex

Description

The invention relates to the field of chemistry and medicine, namely to new compounds capable of delivering nucleic acids to mammalian cells.

Over the past few years, in connection with an increase in the number of genetic diseases, interest in such a field of medicine as gene therapy has increased. Among the methods for delivering genetic material to eukaryotic cells, lipofection is a prominent place - DNA transfer using cationic amphiphiles or liposomes [1]. Cationic amphiphiles and liposomes based on them form electrostatic complexes with nucleic acid molecules that protect nucleic acids from degradation by cellular enzymes, and their penetration into the cell occurs by the mechanism of endocytosis. In vitro delivery of nucleic acids using cationic amphiphiles is a routine routine; however, the in vivo application of this method is limited by low transfer efficiency due to the presence of various types of biological barriers (for example, the interaction of DNA-lipid particles with blood plasma proteins and their reticulum capture endothelial system, as well as low specificity of getting into target organs and tissues) [2]. To increase the efficiency of nucleic acid delivery, special structural elements are introduced into the molecules of cationic amphiphiles, which help them overcome biological barriers [3]. For example, to target specific types of cells, the cationic amphiphilic molecule is modified with peptide or carbohydrate targeted ligands [4].

The cationic amphiphiles include a wide range of chemicals that have common structural features: the presence of positively charged and hydrophobic domains connected by a spacer of various lengths [5-7]. A hydrophilic positively charged domain is necessary for binding to a nucleic acid molecule, and a hydrophobic domain for its encapsulation. Recently, the structure of cationic amphiphiles increasingly includes carbohydrate residues, which act as address markers for the delivery of nucleic acids to special cells and the nucleus [8-14]. In addition, the carbohydrate residue can serve as a basis for the placement of one or more cationic groups [15-17]. Amphiphiles containing carbohydrate residues can cause a pH-dependent transition of amphiphilic aggregates from the lamellar phase to the micellar phase, thereby increasing the efficiency of delivery of genetic material [18]. In addition, the presence of carbohydrate fragments increases the colloidal stability of DNA lipid particles in blood serum [19, 20] and reduces toxicity [21].

The closest in structure to the claimed compounds, the prototype is the cationic amphiphile N- [1- (2,3-dioleloxy) propyl] -N- [2- (6-spermylcarbamoyl) ethyl] -N, N-dimethylammonium trifluoroacetate (DOSPA) [ 22]. The structure of this lipid includes the remainder of 1,2-di-O-oleoyl-rac-glycerol, which forms a hydrophobic domain, the remainder of the natural polycation - spermine - forms a cationic domain, which binds to the phosphate groups of nucleic acids. A disadvantage of the known compound is the low transfection activity when used as an individual compound.

An object of the invention is to obtain new effective synthetic compounds with high transfection activity, low toxicity and capable of delivering nucleic acids to mammalian cells.

The stated technical problem is solved by the proposed compounds, which are carbohydrate-containing cationic amphiphiles of the general formula:

where A is the remainder of 1,2-di-O-tetradecyl-rac-glycerol, B is the remainder of galactose (for (1)), lactose (for (2)) and mannose (for (3)), C is the spermine residue, n = 6, m = 2.

The figure 1 presents the structural formulas of the compounds (1), (2) and (3). The structure of amphiphiles (1), (2) and (3) includes a spermine residue for binding and compaction of a nucleic acid molecule, a 1,2-di-O-tetradecyl-rac-glycerol residue to form a hydrophobic environment. A biodegradable succinyl type linker is used to bind the hydrophobic and spermine moieties. To give carbohydrate residues spatial mobility, monosaccharides are attached to the lipid moieties through hexamethylene bridges.

The new compounds (1), (2) and (3) were obtained according to the general scheme shown in Figure 2. The starting compound in the synthesis was 1-bromo-1-deoxy-2,3-di-O-tetradecyl-rac-glycerol (4) which was reacted with N- (6-hydroxyhexyl) -4-nitrobenzenesulfonamide (5) to give a bifunctional compound (6). The key step in the synthesis of compounds (7a-b) was the glycosylation of compound (6) with 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide, 2,2 ', 3,3', 4 ', 6, 6'-hepta-O-acetyl-β-lactopyranosyl bromide or 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl bromide [23]. To obtain a convenient binding site, the nitro group of the aromatic nucleus was reduced to the amino group using Pd / C as a catalyst. Next, a succinyl spacer was added to amphiphilic molecules (8a-b) by treatment with succinic anhydride to obtain carboxylates (9a-c). The coupling of compounds (9a-c) with N ⁴ , N ⁹ , N ¹² -tri-tert-butoxycarbonyl-1,12-diamino-4,9-diazadodecane was carried out in the presence of O- (1H-benzotriazol-1-yl) -N , N, N ', N'-tetramethyluronium hexafluorophosphate at 4 ° C. At the final stage of synthesis in compounds (10a-c), the amino groups were released by the action of trifluoroacetic acid, and the removal of acetyl protections was 0.04 N. a solution of sodium methylate in methanol.

The synthesis used refined solvents, reagents of domestic (Himmed, Reakhim) and foreign production (Merck, Fluka, Aldrich, Acros). Monitoring the progress of reactions was carried out using TLC. Thin layer chromatography was performed on Kieselgel 60 F ₂₅₄ (Merck), RP-18 F _254S (Merck), Sorbfil (Russia) plates. Spot detection in the chromatograms was carried out with a solution of phosphormolybdenum acid - cerium (IV) sulfate, followed by heating, Dragendorf reagent, potassium permanganate solution and using a UV lamp (254 nm). Column chromatography was performed on silica gel Kieselgel 60 (0.040-0.063 mm); Kieselgel 60 (0.063-0.200 mm), LiChroprep® RP-18 (0.040-0.063 mm, Merck). ¹ H- and ¹³ C-NMR spectra were recorded on a Bruker DPX-300 and Bruker AMX-400 pulsed Fourier spectrometer in CDCl ₃ , a mixture of CDCl ₃ -CD ₃ OD and Py-d5 (internal standard tetramethylsilane). The values of chemical shifts (δ) are given in parts per million (ppm), the spin-spin interaction constants (J) in hertz (Hz). Mass spectra were obtained on a Bruker Ultraflex time-of-flight mass spectrometer (Germany) by matrix laser desorption ionization on a matrix using 2,5-dihydroxybenzoic acid as a matrix. The angles of optical rotation were measured using a Digytor Yasco DIP 360 photoelectric spectropolarimeter (Japan).

To study the ability of the proposed compounds to deliver nucleic acids to mammalian cells, we used a 5-dTACAGTGGAATTGTATGCCTATTA-3 '25-unit oligodeoxyribonucleotide modified at the 3'-end of the fluorescein isothiocyanate (FITC), plasmid DNA (pEGFP-C2, Germany) and 21-stranded double-stranded RNA (siPHK, ICBPM SB RAS) (sense sequence 5'-GCGCCGAGGUGAAGUUCGATT-3 ', antisense strand 5'-UCGAACUUCACCUCGGCGCGG-3'). FITC-ON was synthesized by the phosphitamide method [24] and purified by ion exchange and reverse phase chromatography. The purity of the oligonucleotide was checked by electrophoresis in 15% PAG under denaturing conditions, visualization of the oligonucleotide in the gel was performed using Stains-All staining.

The efficiency of penetration of nucleic acids using cationic amphiphiles into mammalian cells in vitro was studied in experiments on transfection of cells with a FITC-labeled oligonucleotide, plasmid DNA encoding a green fluorescent protein (EGFP) and short interfering RNA directed against a matrix RNA gene encoding EGFP.

A comparative analysis of the claimed compounds with known and widely used compounds, such as Lipofectamine® 2000 and Oligofectamine showed that the proposed compounds have the following advantages.

1) Due to the presence of natural structural modules, including carbohydrate residues, the claimed compounds have low toxic effects on mammalian cells.

2) The claimed compounds have the ability to deliver nucleic acids to mammalian cells, both short and extended, which allows them to be considered as promising agents for transfection of mammalian cells.

3) The inventive compounds deliver nucleic acids to cells in an individual state in the form of aqueous solutions and do not require the use of additional auxiliary lipids.

The inventive compounds do not require a complicated preparation procedure characteristic of liposomal compositions, they are characterized by the ease of preparation of the working solution, for which it is sufficient to dissolve the claimed compound in water or alcohol. In addition, the claimed compounds are stable during storage both in dry form and in the form of concentrated aqueous or alcoholic solutions.

A search in the sources of scientific, technical and patent literature showed that the claimed compounds (1), (2), (3), capable of delivering nucleic acids to mammalian cells, are not described in known sources.

The invention is illustrated by the following examples.

Example 1. Obtaining rac-N- (6-hydroxyhexyl) -N- [2,3-di (tetradecyl-hydroxy) prop-1-yl] -4-nitrobenzenesulfonamide (intermediate compound 6).

To a solution of 0.995 g (1.816 mmol) of rac-1-bromo-1-deoxy-2,3-di-O-tetradecylglycerol (4) in 10 ml of dimethylformamide was added 0.571 g (1.888 mmol) of N- (6-hydroxyhexyl) -4 nitrobenzenesulfonamide (5), cesium carbonate (0.355 g, 1.090 mmol) and tetrabutylammonium iodide (0.067 g, 0.181 mmol). The reaction mixture was stirred for 2 days with heating, was extracted with petroleum ether. The organic extract was evaporated and chromatographed on a silica gel column. The yield of compound (6) was 1.397 g (60%).

Example 2. Obtaining rac-N- [6- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1- yl] -4-nitrobenzenesulfonamide (intermediate 7a).

To a solution of 1.028 g (1.336 mmol) of rac-N- (6-hydroxyhexyl) -N- [2,3-di (tetradecyloxy) prop-1-yl] -4-nitrobenzenesulfonamide (6) in 40 ml of anhydrous benzene was added 0.691 g (4.009 mmol) of calcined cadmium carbonate and boiled in a Soxhlet apparatus. A solution of 1.648 g (4.009 mmol) of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide was added to the reaction mixture. After 2.5 h, the reaction mass was filtered, the solvent was removed in vacuo. The residue was chromatographed on a silica gel column. The yield of compound (7a) was 1.152 g (78%).

Intermediates (7b) and (7c) were obtained as described for the preparation of compound (7a), starting from rac-N- (6-hydroxyhexyl) -N- [2,3-di (tetradecyloxy) prop-1-yl] -4-nitrobenzenesulfonamide (6), 2,2 ', 3,3', 4 ', 6,6'-hepta-O-acetyl-β-lactopyranosyl bromide and 2,3,4,6-tetra-O-acetyl- α-D-mannopyranosyl bromide, respectively. The physicochemical characteristics of compounds (7b) and (7c) correspond to their chemical structure.

Example 3. Obtaining rac-N- [6- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1- yl] -4-aminobenzenesulfonamide (intermediate 8a).

To a solution of 1.126 g (1.024 mmol) rac-N- [6- (2,3,4,6-tetra-O-acetyl- [β-D-galactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy ) prop-1-yl] -4-nitrobenzenesulfonamide (7a) in 14 ml of a mixture of methanol: tetrahydrofuran (2.5: 2), 0.258 g (4.096 mmol) of ammonium formate was added and heated to 60 ° C, a catalytic amount of Pd / C was added. After 30 minutes, the reaction mixture was filtered, and the solvents were removed in vacuo. The residue was chromatographed on a silica gel column. The yield of compound (8a) was 0.933 g (85%).

Intermediates (8b) and (8c) were obtained as described for the preparation of compound (8a), starting from rac-N- [6- (2.2 ', 3.3', 4 ', 6.6'-hepta -O-acetyl-β-lactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1-yl] -4-nitrobenzenesulfonamide (7b) and rac-N- [6- (2,3,4 , 6-tetra-O-acetyl-β-D-mannopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1-yl] -4-nitrobenzenesulfonamide (7c), respectively. The physicochemical characteristics of compounds (8b) and (8c) correspond to their chemical structure.

Example 4. Obtaining rac-N- [6- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1- yl] -4- (3-carboxypropanoylamino) benzenesulfonamide (intermediate 9a).

To a solution of 0.932 g (0.872 mmol) rac-N- [6- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1-yl] -4-aminobenzenesulfonamide (8a) in dichloromethane added 1.025 g (10.23 mmol) of succinic anhydride, 0.024 g (0.201 mmol) of dimethylaminopyridine and 287 μl (2.046 mmol) of triethylamine. After 36 hours, the reaction mixture was washed with an aqueous HCl solution. The organic phase was dried, filtered, and the solvents were removed in vacuo. The residue was chromatographed on a silica gel column. The yield of compound (9a) was 0.747 g (73%).

Intermediates (9b) and (9c) were obtained as described for the preparation of compound (9a), starting from rac-N- [6- (2.2 ', 3.3', 4 ', 6.6'-hepta -O-acetyl-β-lactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1-yl] -4-aminobenzenesulfonamide (8b) and raс-N- [6- (2,3,4 , 6-tetra-O-acetyl-β-D-mannopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1-yl] -4-aminobenzenesulfonamide (8c), respectively. The physicochemical characteristics of compounds (9b) and (9c) correspond to their chemical structure.

Example 5. Obtaining rac-N- [6- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1- yl] -4 - [(N ⁴ , N ⁹ , N ¹² -tri-tert-butoxycarbonyl-12-amino-4,9-diazadodec-1-yl) aminosuccinylamino] benzenesulfonamide (intermediate 10a).

To a solution of 0.642 g (0.549 mmol) of compound (9a) and 0.552 g (1.098 mmol) of N ⁴ , N ⁹ , N ¹² -tri-tert-butoxycarbonyl-1,12-diamino-4,9-diazadodecane, 191 μl were added to dimethylformamide (1.098 mmol) of N, N-diisopropylethylamine and a dropwise solution of 0.416 g (1.098 mmol) of O- (1H-benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate in DMF. After 5.5 hours, chloroform was added to the reaction mass and extracted with an aqueous HCl solution. The organic phase was dried, filtered, and the residue was chromatographed on a silica gel column. The yield of compound (10a) was 0.563 g (62%).

Intermediates (10b) and (10c) were obtained as described for the preparation of compound (10a), starting from compounds (9b) and (9 s), respectively. The physicochemical characteristics of compounds (10b) and (10c) correspond to the chemical structure.

Example 6. Preparation of tris (trifluoroacetate) rac-N- [6- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1-yl] -4 - [(12-amino-4,9-diazadodec-1-yl) aminosuccinylamino] benzenesulfonamide (intermediate 11a).

To a solution of 0.669 g (0.404 mmol) of compound (10a) in dichloromethane was added 4.416 ml (19.8 mmol) of trifluoroacetic acid. After 3.5 hours, the solvents were removed in vacuo. The residue was chromatographed on a silica gel column. The yield of compound (11a) was 0.472 g (76%).

Intermediates (11b) and (11c) were prepared as described for the preparation of compound (11a) based on (10b) and (10c), respectively. The physicochemical characteristics of compounds (11b) and (11c) correspond to their chemical structure.

Example 7. Obtaining trihydrochloride rac-N- [6- (β-D-galactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1-yl] -4 - [(12-amino-4, 9-diazadodec-1-yl) -aminosuccinylamino] benzenesulfonamide (1).

To 0.472 g (0.278 mmol) of compound (11a), 0.04 N was added. a solution of sodium methylate in methanol. After 1 h, a solution of 4 N was added to the reaction mass. HCl in dioxane until the pH is 5. The solvent was removed in vacuo and the residue was chromatographed on a column. The resulting product was dissolved in 7 ml of distilled water and dialyzed against water in the membrane. After lyophilization, the yield of compound (1) was 0.199 g (55%). ¹ H-NMR (δ, ppm): 0.65 (t, 6H, J = 6.5, 2CH ₂ C H ₃ ), 1.05 (br s, 44H, 2 (CH ₂ ) ₁₁ ), 1.13-1.26 ( m, 4H, CH ₂ CH ₂ ), 1.31-1.49 (m, 8H, 3C H ₂ CH ₂ O, C H ₂ CH ₂ N), 1.68-1.95 (m, 6H, spermine protons), 2.28-2.40 (m , 2H) and 2.58-2.69 (m, 2H, NCOCH ₂ CH ₂ COO), 2.81-3.55 (m, 24H, CHH _a O, OCH ₂ CH, 2CH ₂ N, 2CH ₂ O, spermine protons), 3.74-3.95 m, 4H, H5-Gal, H _ab 6-Gal, CHH _b O), 4.11-4.20 (m, 2H, H2-Gal, H3-Gal), 4.35-4.31 (m, 1H, H4-Gal), 4.31 -4.49 (d, 1H, J = 7.6, H1-Gal), 7.78-7.86 (m, 2H) and 8.01-8.09 (m, 2H, Ar), 8.95-9.02 (br.s, 1H, NH). ¹³ C-NMR (δ, ppm): 14.21, 22.87, 23.94, 24.21, 25.56, 25.97, 26.49, 26.81, 27.18, 28.34, 29.55, 29.76, 29.92, 30.11, 30.61, 30.96, 32.06, 32.60, 36.24 , 37.78, 39.00, 45.19, 45.54, 47.26, 50.11, 62.19, 69.52, 70.13, 70.55, 71.39, 71.69, 72.46, 75.13, 76.67, 78.75, 105.08, 128.78, 172.23, 174.08. Mass spectrum (MALDI-TOF), m / z: 1185.864 [M-3HCl + H] ⁺ , 1207.847 [M-3HCl + Na] ⁺ , 1223.825 [M-3HCl + K] ⁺ calculated for C ₆₃ H ₁₂₀ N ₆ O ₁₂ S: 1184.8685.

Compounds (2) and (3) were obtained as described for the preparation of compound (1) starting from (11b) and (11c), respectively. The physicochemical characteristics of compounds (2) and (3) correspond to their chemical structure.

Rac-N- [6- (β-lactopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1-yl] -4 - [(12-amino-4,9-diazadodec-1-) trihydrochloride il) aminosuccinylamino] benzenesulfonamide (2).

The yield of compound (2) was 64%. [α] _D ³³ -3.07 (s0.8, CHCl ₃ -CH ₃ OH, 1: 5). ¹ H-NMR (δ, ppm): 0.81 (t, 6H, J = 6.8, 2CH ₂ C H ₃ ), 1.16-1.28 (m, 48H, 2 (CH ₂ ) ₁₁ , (CH ₂ ) ₂ ); 1.36-1.66 (m, 20H, 3C H ₂ CH ₂ O, 5C H ₂ CH ₂ N, CH ₂ CH ₂ ), 3.18-3.57 (m, 12H, 2NC H ₂ , 2OCH ₂ C H ₂ , CH ₂ O, CHH _a O, CH ₂ C H CH ₂ ), 3.65-3.75 (m, 2H, CHH _b O, H5-Lac), 3.78-3.86 (m, 1H, H5'-Lac), 3.91-4.12 (m, 4H , H _ab 6-Lac, H _ab 6'-Lac), 4.37 (d, 1H, J = 7.8, H-1 'Lac), 4.41 (m, 1H, H-4 Lac), 4.43 (d, 1H, J = 7.8, H-1 Lac), 4.79 (dd, 1H, J = 7.8, 9.4, H-2 Lac), 4.89 (dd, 1H, J = 3.4, 10.3, H-3 'Lac), 5.03 (dd , 1Н, J = 7.8, 10.3, Н-2 'Lac), 5.12 (t, 1Н, J = 9.4, Н-3 Lac), 5.29 (m, 1Н, Н-4' Lac), 7.75-7.92 (m 4H, Ar). ¹³ C-NMR (δ, ppm): 14.84, 23.48, 24.28, 25.50, 26.40, 27.29, 28.54, 30.16, 30.55, 30.72, 30.72, 31.23, 33.63, 38.47, 46.04, 46.47, 48.05, 50.46, 62.32 , 62.49, 70.27, 70.40, 71.24, 72.28, 72.88, 75.25, 76.79, 77.49, 78.72, 79.52, 82.04, 104.51, 105.80, 120.28, 129.27, 134.58, 144.82, 172.96, 174.35. Mass spectrum (MALDI-TOF), m / z: 1347.700 [M-3HCl + H], calculated for C ₆₉ H ₁₁₃₀ N ₆ O ₁₇ S: 1346.9213.

Rac-N- [6- (β-D-mannopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1-yl] -4 - [(12-amino-4,9-diazadodec- trihydrochloride trihydrochloride 1-yl) aminosuccinylamino] benzenesulfonamide (3).

The yield of compound (3) was 55%. [α] _D ²³ 6.48 (c1, CH ₃ OH). ¹ H-NMR (δ, ppm): 0.67 (t, 6H, J = 6.7, 2 CH ₂ C H ₃ ), 1.10 (br.s, 44H, 2 (CH ₂ ) ₁₁ ), 1.15-1.25 (m, 4H, CH ₂ CH ₂ ), 1.34-1.45 (m, 8H, 3C H ₂ CH ₂ O, C H ₂ CH ₂ N, 1.68-1.95 (m, 6H, spermine protons), 2.28-2.40 (m , 2H) and 2.58-2.69 (m, 2H, NCOCH ₂ CH ₂ COO), 2.81-3.55 (m, 24H, CHH _a O, OCH ₂ CH, 2CH ₂ N, 2CH ₂ O, spermine protons), 3.61 (dt , J = 9.6, 6.4, 1H, CHH _b O), 3.81-3.85 (m, 2H, H5-Man), 4.04 (dd, 1H, J = 2.4, 12.2, H a 6-Man), 4.23 (dd, 1H, J = 5.2, 12.2, H _b 6-Man), 4.73 (d, 1H, J = 1.6, H1-Man), 5.16 (dd, 1H, J = 1.6, 3.1, H2-Man), 5.19 (dd , 1H, J = 9.2, 9.8, H4-Man), 5.28 (dd, 1H, J = 3.1, 9.8, H3-Man), 7.75-7.91 (m, 2H) and 8.05-8.15 (m, 2H, Ar) 8.95-9.02 (m, 1H, NH), 11.05-11.45 (m, 1H, NH). ¹³ C-NMR (δ, ppm): 24.67, 25.75, 26.33, 27.65, 29.39, 29.62, 29.71, 29.77, 30.46, 31.24, 31.89, 45.32, 46.75, 47.19, 61.93, 66.91, 67.11, 67.99, 70.46, 71.50, 71.78, 72.32, 78.81, 100.77, 119.43, 1 22.56, 123.92, 128.50, 134.00, 135.98, 143.97, 148.84, 172.21, 173.78. Mass spectrum (MALDI-TOF), m / z: 1185.499 [M-3HCl + H] ⁺ , 1207.483 [M-3HCl + Na], 1223.451 [M-3HCl + K] ⁺ calculated for C ₆₃ H ₁₂₀ N ₆ O ₁₂ S: 1184.8685.

Example 8. The effect of carbohydrate-containing cationic amphiphile (1) on the viability of BHK, BHK IR-780, HeLa, HEK 293 cells.

BHK cell lines (Syrian hamster kidney embryonic cells), BHK IR-780 (modified Syrian hamster kidney embryonic cells), HEK 293 (human embryonic kidney cells) were cultured in DMEM medium containing 10% fetal calf serum in an atmosphere of 5% -CO ₂ at 37 ° C.

Cell viability after incubation with cationic amphiphile (1) was determined using the MTT test, which is based on the ability of living cells to convert tetrazole-based compounds (MTT) into brightly colored formazan crystals, which allows spectrophotometrically to estimate the number of living cells in the preparation. For this, cells were plated in 96-well plates (0.1 × 10 ⁵ cells per well for HEK 293 cells and 0.03 × 10 ⁵ cells per well for BHK, BHK IR-780 cells). After 24 hours, the medium was changed in the wells and a solution of cationic amphiphile (1) in DMEM medium was added to the cells to a final concentration in the well of 1 to 80 μM. Cells were incubated in the presence of amphiphile for 24 hours under the same conditions. At the end of incubation, MTT solution (5 mg / ml) in phosphate-buffered saline was added to the cells without changing the medium to a concentration of 0.5 mg / ml and incubated for 3 h under the same conditions. The medium was removed, 100 μl of dimethyl sulfoxide, in which the formazan crystals formed in the cells were dissolved, were added to the cells, and the optical density was measured on a multichannel spectrophotometer at wavelengths of 570 and 630 nm, where A ₅₇₀ is the absorption of formazan and A ₆₃₀ is the background of the cells.

From the experimental data, the IC ₅₀ value, the concentration of the compound at which the death of 50% of the cells is observed, was calculated. The IC ₅₀ values of cationic amphiphilic (1) for cells are shown in table 1.

Table 1 Cells The presence of 10% serum in the growth medium IC ₅₀ μm HEK 293 + > 40 - 25 BHK IR-780 + > 40 - eighteen BHK + > 40 - 17

From the above data it is seen that treatment of cells with cationic amphiphile (1) causes their effective death only at concentrations of compounds above 17 μM in the absence of serum in the growth medium, and in the presence of serum above 40 μM, which indicates a low toxicity of the claimed compound.

Example 9. Penetration into cells of a FITC-labeled oligonucleotide using cationic amphiphile (1).

The study of the penetration of a FITC-labeled oligonucleotide into HEK 293 cells was carried out using flow cytometry. Delivery efficiency was evaluated by the number of transfected cells of the total number of cells in the sample. Cells were planted in 24-well plates (2 × 10 ⁵ cells per well) and cultured for one day at 37 ° C. in an atmosphere containing 5% CO ₂ . Before transfection for experiments in the presence or absence of serum, the medium in the wells was replaced with 200 μl of DMEM medium containing 10% FBS or DMEM without serum, respectively. A solution of cationic amphiphile (1) (10 μM) in OptiMEM medium was mixed with a solution of FITC-labeled oligonucleotide (5 μM) in the same medium, the resulting complexes were added to the cells and incubated for 4 hours. After incubation, the cells were washed with PBS (300 μl ), 50 μl of trypsin solution was added and incubated for 1-2 min (37 ° C, 5% CO ₂ ). At the end of the incubation, 200 μl of DMEM with 10% serum was added to the wells, the cells were suspended and transferred to tubes. The resulting cell suspension was centrifuged at 1000-1200 rpm, 4 ° C, the medium was taken and washed with 1 ml of PBS. Then the cells were fixed with 500 μl of a 2% solution of formaldehyde in PBS. Analysis of the level of cell transfection was carried out on a BD FACSAria flowmeter (Becton Dickinson). In these experiments, the number of cells transfected with a FITC-labeled oligonucleotide and the average level of cell fluorescence determined at an excitation wavelength of 488 nm were determined (the device uses a coherent sapphire laser (20 mV)).

The results of the delivery of BHK cells to a FITC-labeled oligonucleotide (5 μM) with cationic amphiphile (1) (5, 10 μM) in the presence or absence of serum in the growth medium are shown in Table 2.

table 2 The concentration of lipid, μm The presence of 10% serum in the growth medium The number of transfected cells,% 5 - 61,4 + 94.6 10 - 63.3 + 94.6

It can be seen from the above data that in the presence of amphiphile (1), the FITC-labeled oligonucleotide efficiently (the number of transfected cells exceeds 60%) penetrates into the cells in the absence of serum in the growth medium. The presence of 10% serum in the growth medium significantly increases the efficiency of transfection: the number of transfected cells in this case approaches the maximum possible.

Example 10. Transfection of HEK 293 cells with plasmid DNA using cationic amphiphile (1).

Plasmid DNA was delivered to HEK 293 cells by flow cytometry. Transfection efficiency was evaluated by the number of cells containing green fluorescent protein (EGFP) of the total number of cells in the sample. Cells were plated in 24-well plates (2 × 10 ⁵ cells per well for HEK 293 cells) and cultured overnight at 37 ° C. in an atmosphere containing 5% CO ₂ . Before transfection for experiments in the presence or absence of serum, the medium in the wells was replaced with 200 μl of DMEM medium containing 10% FBS or DMEM without serum, respectively. A solution of compound (1) (10 μM) in OptiMEM medium was mixed with a solution of pEGFP-C2 (0.3 μg / μl) in the same medium, the resulting mixture was added to the cells and incubated for 24 hours. After 4 hours in wells with serum-free medium replaced the medium with DMEM with 10% serum. At the end of the incubation, the cells were processed and analyzed as described in Example 9. Table 3 presents the results of transfection of plasmid DNA cells with pEGFP-C2 with cationic amphiphile (1).

Table 3 Cells The presence of 10% serum in the growth medium The number of transfected cells,% HEK 293 - 30.3 + 2.2

From the above data it is seen that using cationic amphiphilus (1), plasmid DNA penetrates into cells in a noticeable amount in the absence of serum in the growth medium.

Example 11. Transfection of BHK IR-780 cells with short interfering RNA using cationic amphiphile (1).

A study of siRNA penetration aimed at suppressing the synthesis of green fluorescent EGFP protein was carried out on BHK line IR-780 cells stably expressing this protein. As a target, mRNA encoding an EGFP protein was chosen, thus, by reducing the fluorescence of cells determined by this protein, one can judge the efficiency of siRNA delivery into the cytoplasm.

Cells were plated in 24-well plates (0.2 × 10 ⁵ cells per well for BHK IR-780 cells) and cultured for one day at 37 ° C in an atmosphere containing 5% CO ₂ . Before transfection for experiments in the presence or absence of serum, the medium in the wells was replaced with 200 μl of DMEM medium containing 10% FBS or DMEM without serum, respectively. A solution of cationic amphiphile (1) (10 μM) in OptiMEM medium was mixed with a siRNA solution (50 nM) in the same medium, the resulting complexes were added to the cells and incubated for 72 hours. After 4 hours, the medium in wells with serum-free medium was replaced with DMEM with 10% serum. At the end of the incubation, the cells were processed as described in Example 9. Table 4 presents the results of transfection of cells with short interfering RNA with cationic amphiphile (1), determined by the level of decrease in EGFP protein expression.

Table 4 The concentration of lipid, μm The presence of 10% serum in the growth medium Suppression of EGFP Expression,% 10 - 81.1 + 14.2

It can be seen from the above data that cationic amphiphile (1), in an amount that is not toxic to cells, promotes highly efficient penetration of siRNA into cells, as evidenced by 80% inhibition of the expression of a given protein in the absence of serum in the growth medium.

Thus, the above examples clearly indicate the ability of the proposed carbohydrate-containing cationic amphiphiles to effectively facilitate the penetration of nucleic acids into mammalian cells, which allows them to be used as agents for the delivery of nucleic acids to mammalian cells.

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Claims (1)

Carbon-containing polycationic amphiphiles, which are rac-N- [6- (β-D-glycopyranosyloxy) hexyl] -N- [2,3-di (tetradecyloxy) prop-1-yl] -4 - [(12-amino) trihydrochlorides 4,9-diazadodec-1-yl) amino-succinylamino] benzenesulfonamide of the general formula:

where A is the residue of 1,2-di-O-tetradecyl-rac-glycerol, B is the remainder of galactose (for (1)), lactose (for (2)) and mannose (for (3)), C is the spermine residue, n = 6, m = 2,
with the ability to deliver nucleic acids to mammalian cells.

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