RU2233834C2 - Cationic dimeric amphiphiles as agents for transfection and method for their preparing - Google Patents

Abstract

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention describes dimeric amphiphiles of the general formula (I) $$$ wherein R₁ and R₂ are similar or can differ of one another and mean saturated or unsaturated hydrocarbon (C₁₂-C₂₂)-chain; R₃ and R₄ are similar or can differ of one another and mean lower alkyl; R₅ can mean the following values: saturated (C₃-C₂₂)-chain; residue of mono-, di- or triethylene glycol, cystine residue; n and m are similar or can differ of one another and mean a whole number from 1 to 3; X' and Y; are similar or can differ of one another and mean nontoxic anions used as agents for transfection. Invention describes also a method for preparing such compounds that involves alkylation of free amino-group in amino acid diether to tertiary amino-group by interaction with formalin in the presence of 80-85% formic acid and binding two molecules of diether using active derivative of the corresponding spacer in medium of polar aprotonic organic solvent.

EFFECT: improved preparing method.

3 cl, 7 ex

Description

The invention relates to new chemical compounds, specifically to compounds of General formula I

where R ₁ and R _{2 are the} same or may differ from each other and mean a saturated or unsaturated hydrocarbon chain C _12-22 ; R ₃ and R _{4 are the} same or may differ from each other and mean lower alkyl; R ₅ may have the following meanings: saturated C _3-22 chain; the residue of mono-, di- or triethylene glycol; cystine residue; n and m are the same or may differ from each other and mean an integer from 1 to 3; X'and Y 'are the same or may differ from each other and mean non-toxic anions, which are physiologically active compounds and can be used as transfection agents.

These compounds, their properties and the preparation method are not described in the literature.

Currently, cationic amphiphiles of both lipid and non-lipid nature, and liposomes based on them are widely used as transfection agents for the delivery of therapeutic genes to eukaryotic cells. This process was developed due to the simplicity and accessibility of the starting reagents, the ability to transfer plasmid DNA of virtually unlimited size and protect it from inactivation by the action of cell enzymes. Today, the transfer of nucleic acids using cationic liposomes is a fairly developed and widely used method both in vitro, ex vivo and in vivo. It should be noted that cationic liposomes possess such important properties as non-toxicity, non-immunogenicity and non-infectivity, they are stable during storage and economically available [Lasic D., Liposome in gene delivery, CRC Press, Boca Raton, 1997].

Positively charged derivatives of diglycerides and cationic liposomes based on them are known as transfection agents [Questions of medical chemistry, 1997, v. 43, pp. 3-10].

Bifunctional surface agents containing two quaternary ammonium groups are described [Chem. Commun., 1997, p. 2287-2288].

The antimicrobial properties of dimeric molecules were investigated; it turned out that the compounds have a higher activity compared to monomeric amphiphiles and are effective for the destruction of many gram-negative and gram-positive microorganisms [J. Chem. Soc., Perkin Trans. 2, 1994, p. 1871-1875]. Bisquaternary ammonium salts can be successfully used as phase transfer catalysts in reactions with inorganic or organic dianions [Tetrahedron Letters, 1989, 30 (13), 1683-1686]. However, the use of dimeric bis-quaternary ammonium salts as transfection agents is not described.

Known compounds are prepared by reacting 1-bromo-2,3-di-O-acyl-propanediol with dimethylamine in ethanol under heating and pressure, followed by quaternization with dibromoalkanes in boiling ethanol.

The aim of the invention are new dimeric amphiphiles of the general formula I, which can be used as transfection agents.

Cationic dimeric amphiphiles of formula I are prepared according to a scheme which consists in the alkylation of the free amino group of amino acid diesters to a tertiary amino group by reaction with formalin in the presence of 80-85% formic acid and the binding of two diester molecules using the active derivative of the corresponding spacer in a polar aprotic organic solvent ( dimethylformamide, dimethyl sulfoxide, acetonitrile).

The use of a mixture of formalin with 80% formic acid in the reaction instead of a mixture of paraforms - 99% formic acid [Weigand-Hilgetag. Experimental methods in organic chemistry. - M .: Chemistry, 1964, p.489] avoids racemization and obtain pure optically active compounds.

The reaction of binding of two diester molecules in high-boiling aprotic organic solvents increases the yield of the target products to 70-75% compared with the reaction in ethanol (~ 40%).

The structure of the compounds obtained is proved by IR, PMR spectroscopy and mass spectrometry.

The new compounds have low toxicity, are capable of efficient transfection, their use is not associated with pre-treatment of cells to enhance the permeability of the cell membrane, they can be used in vivo.

The following examples illustrate the invention.

Example 1

Preparation of N, N’-bis (1,4-dihexadecyl-N, N-dimethyl-L-asparaginyl) -1,3-propane diiodide.

While cooling to 0 ° C, 1.55 ml (18 mmol) of formalin, 1.36 ml (30 mmol) of 85% formic acid, 2.5 g (6 mmol) of L-aspartic acid dihexadecyl ester are placed in a round-bottom flask and heated at temperature of 70 ° C for 10 hours. Then, the reaction mixture was diluted with 50 ml of chloroform and washed with 100 ml of 0.1N NaHCO ₃ solution and water to pH 7. The chloroform layer was dried and evaporated in vacuo.

Obtain 2.5 g (93%) of dihexadecyl ether of N, N-dimethyl-L-aspartic acid, so pl. 47-48 ° C, [α] D ²⁰ +7 (c = 1, CCl ₄ ).

IR spectrum (liquid paraffin, ν _max , cm ^-1 2900 (СН ₃ ), 2830 (СН ₂ ), 1730 (СО), 1460 (СН ₃ ), 1365 (СН ₃ ), 1155 (NC), 1090 (С -O), 1020 (C-O), 780, 720, 640.

1 H-NMR spectrum (CDC13, δ, ppm): 0.80 (t, 6H, CH ₃ ), 1.20 (s, 52H, CH ₂ ), 1.54 (t, 4H, βCH ₂ CH ₂ OCO), 2.2 (s, 6H, (CH ₃ ) ₂ ), 2.7 (m, 2H, CH ₂ CH), 4.05 (t, 4H, αCH ₂ OCO), 4.86 (m, 1H, C * H).

Mass spectrum: [M +] 609.5.

To a solution of 0.3 g (0.493 mmol) of N, N-dimethyl-L-aspartic acid dihexadecyl ester in 3 ml of absolute ethanol is added a solution of 0.061 g (0.205 mmol) of 1,3-diiodopropane in 3 ml of absolute ethanol. The mixture is boiled in an oil bath for 48 hours. Next, the reaction mass is evaporated and 3 ml of ether are added. The precipitate was centrifuged for 15 minutes at 7 thousand rpm.

0.11 g (36%) of N, N’-bis (1,4-dihexadecyl-N, N-dimethyl-L-asparaginyl) 1,3-propane diiodide is obtained, mp 98-104 ° C.

Similarly, when carrying out the reaction in dimethyl sulfoxide with heating to 100 ° C for 36 hours, 0.21 g (70%) of N, N'-bis (1,4-dihexadecyl-N, N-dimethyl-D-asparaginyl) 1 is obtained, 3-propane diiodide.

IR spectrum (liquid paraffin, ν _max , cm ^-1 ): 2920 (СН ₃ ), 2830 (СН ₂ ), 1730 (С = O), 1470 (СН ₂ ), 1380 (СН ₃ ), 1150 (NC) 1090 (C-O); 1020 (C-O); 780, 725, 635.

1 H-NMR spectrum (CDC1 ₃ , δ, ppm): 0.85 (t, 12H, CH ₃ ), 1.20 (s, 104H, CH ₂ ), 1.6 (m, 10H, βCH ₂ ) 3.1 (m, 4H, CH-CH ₂ ), 3.5 (t, 4H, N-CH ₂ ), 3.58 (s, 12H, N ⁺ (CH ₃ ) ₂ ), 4.1 ( t, 4H, αCH ₂ OCO), 4.2 (t, 4H, αCH ₂ OCO), 4.7 (t, 2H, C * H).

Mass spectrum: [M +] 1261.0.

Similarly, N, N ^' -buc (1,4-dihexadecyl-N, N-dimethyl-L-asparaginyl) 1,7-heptane diiodide is obtained.

Yield 72%, mp 102-106 ° C.

1 H-NMR spectrum (CDCl ₃ , δ, ppm): 0.85 (t, 12H, CH ₃ ), 1.20 (s, 110H, CH ₂ ), 1.6 (m, 12H, βCH ₂ ) 3.1 (m, 4H, CH-CH ₂ ), 3.5 (t, 4H, N-CH ₂ ), 3.58 (s, 12H, N ⁺ (CH ₃ ) ₂ ), 4.1 ( t, 4H, αCH ₂ OCO), 4.2 (t, 4H, αCH ₂ OCO), 4.7 (t, 2H, C * H).

Mass spectrum: [M +] 1317.1.

Similarly, N, N’-bis (1,4-dihexadecyl-N, N-di-N, N-dimethyl-L-asparaginyl) 1,11-undecane diiodide is obtained.

Yield 69%, mp 92-96 ° C.

1 H-NMR spectrum (CDCl ₃ , δ, ppm): 0.85 (t, 12H, CH ₃ ), 1.20 (s, 118H, CH ₂ ), 1.6 (m, 12H, βCH ₂ ) 3.1 (m, 4H, CH-CH ₂ ), 3.5 (t, 4H, N-CH ₂ ), 3.58 (s, 12H, N (CH ₃ ) ₂ ), 4.1 (t 4H, αCH ₂ OCO), 4.2 (t, 4H, αCH ₂ OCO), 4.7 (t, 2H, C * H).

Mass spectrum: [M +] 1373.3.

Example 2

Preparation of N, N’-bis (1,4-dihexadecyl-N, N-dimethyl-L-glutamyl) 1,3-propane.

When cooled to 0 ° C, 0.9 ml (22 mmol) of formalin, 1.42 ml (36 mol) of 80% formic acid, 4.4 g (8 mmol) of L-glutamic acid dihexadecyl ester are placed in a round-bottom flask and heated at temperature of 70 ° C for 10 hours. Then the reaction mass is diluted with 50 ml of chloroform and washed with 100 ml of 0.1N NaHCO ₃ solution and water to pH 7.

The chloroform layer was dried and evaporated in vacuo. Obtain 3.7 g (82%) of dihexadecyl ester of N, N-dimethyl-L-glutamic acid, so pl. 34-37 ⁰ C, [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ +9 ⁰ (c = 1, CCl ₄ ).

IR spectrum (liquid paraffin, ν _max , cm ^-1 ): 2920 (СН ₃ ), 2835 (СН ₃ ), 1730 (С = O), 1465 (СН ₂ ), 1370 (СН ₃ ), 1155 (NC) 1090 (C-O); 1020 (C-O); 780, 710, 640.

1 H-NMR spectrum (CDC13, δ): 0.80 (t, 6H, CH ₃ ), 1.20 (br s, 52H, CH ₂ ), 1.54 (t, 4H, CH ₂ CH ₂ OCO), 1.95 (m, 2H, δ CH ₂ ), 2.26 (s, 6H, N (CH ₃ ) ₂ ), 3.15 (m, 1H, β CH ₂ ), 3.6 (m, 1H, β CH ₂ ), 4.05 (t, 4H, CH ₂ OCO), 4.86 (m, 1H, CH).

Mass spectrum: [M +] 623.3.

To a solution of 0.25 g (0.4 mmol) of N.N-dimethyl-L-glutamic acid dihexadecyl ester in 3 ml of dimethylformamide was added a solution of 0.04 g (0.1 mmol) of 1,3-diiodopropane in 3 ml of dimethylformamide. The mixture is heated in an oil bath at a temperature of 100 ° C for 48 hours. Next, 3 ml of ether is added to the reaction mass. The precipitate formed is filtered off. Obtain 0.14 g (70%) of N, N’-bis (1,4-dihexadecyl-N, N-dimethyl-L-glutamyl) 1,3-propane diiodide, mp 88-96 C.

IR spectrum (liquid paraffin. Ν _max , cm ^-1 ) 2920 (СН ₃ ), 2825 (СН ₂ ), 1730 (С = 0), 1460 (СН2), 1360 (СН ₃ ), 1155 (NC), 1090 (C-0), 1020 (C-0), 780, 715, 635.

1 H-NMR spectrum (CDCl ₃ , δ): 0.80 (t, 12H, CH ₃ ), 1.20 (br s, 104H, CH ₂ ), 1.6 (m, 10H, βCH ₂ ), 1, 6 (m, 2H, NCH ₂ -CH ₂ ), 2.15 (m, 2H, CH-CH ₂ ), 2.48 (t, 4H, OCOCH ₂ ), 3.57 (t, 4H, N-CH ₂ ), 3.58 (s, 12H, N ⁺ (CH ₃ ) ₂ ), 4.1 (t, 4H, CH ₂ OCO), 4.2 (t, 4H, CH ₂ OCO), 4.55 ( m, 2H, CH).

Mass spectrum: [M +] 1288.7.

Similarly, N, N’-bis (1,4-dihexadecyl-N, N-dimethyl-L-glutamyl) 1, 7-heptane diiodide is obtained.

Yield 70%, mp. 94-100 ° C.

1 H-NMR spectrum (CDCl ₃ , δ): 0.80 (t, 12H, CH ₃ ), 1.20 (br s, 110H, CH ₂ ), 1.6 (m, 16H, βCH ₂ ), 2, 15 (m, 2H, CH-CH ₂ ), 2.48 (t, 4H, OCOCH ₂ ), 3.56 (t, 4H, N-CH ₂ ), 3.58 (s, 12H, K (CH ₃ ) ₂ ), 4.1 (t, 4H, CH ₂ OCO), 4.2 (t, 4H, CH ₂ OCO), 4.55 (m, 2H, CH).

Mass spectrum: [M +] 1344.8.

Similarly, N, N’-bis (1,4-dihexadecyl-N, N-dimethyl-L-glutamyl) 1, 11-undecane diiodide is obtained.

Yield 75%, mp. 80-88 ° C.

1 H-NMR spectrum (CDCl ₃ , δ): 0.80 (t, 12H, CH ₃ ), 1.20 (br s, 118H, CH ₃ ), 1.6 (m, 16H, βCH ₂ ), 2, 15 (m, 2H, CH-CH ₂ ), 2.47 (t, 4H, OSOOCH ₂ ), 3.55 (t, 4H, N-CH ₂ ), 3.57 (s, 12H, N ⁺ (CH ₃ ) ₂ ), 4.1 (t, 4H, CH ₂ OCO), 4.2 (t, 4H, CH ₂ OCO), 4.55 (m, 2H, CH).

Mass spectrum: [M +] 1400.9.

Example 3

Preparation of N, N’-bis (1,4-dioleyl-N, N-dimethyl-L-glutamyl) 1,3-propane diiodide.

When cooled to 0 ° C, 0.4 ml (10.5 mmol) of 80% formic acid and 0.54 ml (6.3 mmol) of formalin are placed in a round-bottom flask. 1.35 g (2.1 mmol) of L-glutamic acid dioleyl ester is then added and heated in an oil bath at 70 ° C. for 5 hours. The reaction mass is diluted with 50 ml of chloroform and washed with 400 ml of 0.1N NaHCO ₃ solution and water to pH 7. The chloroform layer is dried and evaporated in vacuo. 0.9 g (64%) of N, N-dimethyl-L-glutamic acid dioleic ester is obtained in the form of a thick oil.

IR spectrum (ν _max , cm ^-1 ): 2920 (СН ₃ ), 2835 (СН ₂ ), 1735 (С = O), 1656, (СН = СН), 1465 (СН ₂ ), 1365 (СН ₃ ) 1165 (NC), 1090 (C-O), 1020 (C-O), 720.

1 H-NMR spectrum (DCCl ₃ , δ, dm): 0.805 (t, 6H, CH ₃ ), 1.3 (s, 44H, CH ₂ ), 1.6 (m, 6H, βCH ₂ ), 2 , 0 (q, 8H, CH ₂ CH =), 2.41 (d, 6H, H (CH ₃ ) 2), 2.75 (t, 2H, CH ₃ CO), 3.3 (m, 1H, CH ₂ CH ₂ ), 3.6 (m, 1H, CH ₂ CH), 4.0 (t, 4H, αCH ₂ ), 4.3 (m, 1H, C * H), 5.3 (t, 4H, CH = CH).

Mass spectrum: [M +] 675.0.

To a solution of 0.15 g (0.22 mmol) of N, N-dimethyl-L-glutamic acid dioleic ester 5 in 1 ml of dimethylformamide was added 0.03 g (0.11 mmol) of 1,3-diiodopropane. The mixture is heated in an oil bath for 56 hours at a temperature of 100 ° C. Next, the reaction mass is evaporated and 1 ml of methanol is added. The solution is washed with hexane, the layers are separated, the methanol layer is evaporated. 0.091 g (65%) of N, N’-bis (1,4-dioleyl-N, N-dimethyl-L-glutanyl) 1,3-propane diiodide is obtained.

1 H-NMR spectrum (DCCl ₃ , δ, dm): 0.85 (t, 12H, CH ₃ ), 1.25 (s, 88H, CH ₂ ), 1.57 (m, 12H, βCH2), 2.0 (t, 16H, CH ₂ CH =), 2.7 (t, 2H, CH ₂ CO), 3.0 (t, 4H, N-CH ₂ ), 3.42 (s, 12H, N ⁺ (CH ₃ ) ₂ ), 4.0 (t, 8H, OCCH ₂ ), 4.1 (m, 2H, CH ₂ CH ₂ ), 4.2 (m, 2H, CH ₂ CH), 4.3 (m, 2H, C * H); 5.3 (t, 8H, CH = CH).

Mass spectrum: [M +] 1392.0.

Example 4. Obtaining N, N’-bis- (1,4-dihexadecyl-N, N-dimethyl-L-glutanyl) -2,2-ethylene glycol dichloride.

To a solution of 0.15 g (0.24 mmol) of N, N-dimethyl-L-glutamic acid dihexadecyl ester in 1 ml of dimethylformamide, 0.01 g (0.1 mmol) of 2,2-dichloroethylene glycol is added. The mixture is heated in an oil bath for 56 hours. At a temperature of 100 ° C. Next, the reaction mass is evaporated and 1 ml of methanol is added. The solution is washed with hexane, the layers are separated, the methanol layer is evaporated. Obtain 0.086 g (65%) of N, N’-bis- (1,4-dihexadecyl-N, N-dimethyl-L-glutanyl) -2,2-ethylene glycol dichloride.

IR spectrum (liquid paraffin, ν _max , cm ^-1 2920 (СН ₃ ), 2825 (СН ₂ ), 1730 (С = O), 1460 (СН ₂ ), 1360 (СН ₃ ), 1155 (NC), 1090 (C-O), 1020 (C-O), 780, 715, 635.

1 H-NMR spectrum (CDCl ₃ , δ): 0.80 (t, 12H, CH ₃ ), 1.20 (br s, 104H, CH ₂ ), 1.6 (m, 10H, βCH ₂ ), 2, 2 (m, 2H, CH-CH ₂ , 4H, CH ₂ O), 2.48 (t, 4H, OCOCH ₂ ), 3.57 (t, 4H, N-CH ₂ ), 3.58 (s, 12H, N ⁺ (CH ₃ ) ₂ ), 4.1 (t, 4H, CH ₂ OCO), 4.2 (t, 4H, CH ₂ OCO), 4.55 (m, 2H, CH).

Mass spectrum: [M +] 1288.7.

Example 5

Preparation of 1,1’-bis - [(1,4-dihexadecyl) -L-glutamyl] N, N-bis- (trimethyl) cystine diiodide.

To a solution of 0.3 g (0.46 mmol) of 1,1'-bis- (hydroxysuccinyl) -N, N'-tert-butoxycarbonyl) cystine in 2 ml of anhydrous ethyl acetate is added a solution of 0.55 g (0.92 mmol) of dihexadecyl ether L-glutamic acid. The resulting solution was kept at room temperature for 48 hours. The solvent was removed in vacuo. The resulting product was recrystallized from acetonitrile. 0.48 g (68%) of 1,1′-bis - [(1,4-dihexadecyl-L-glutamyl] -, N, N’-bis- (tert-butyloxycarbonyl) cystine, mp 54 ° C, are obtained.

IR spectrum (liquid paraffin, ν _max , cm ^-1 ): 2920 (СН ₃ ), 2835 (СН ₂ ), 1735 (С = O), 1680 (1st amide band), 1515 (2nd amide lane), 1465 (CH ₂ ), 1370 (CH ₃ ), 1170 (C-0), 765, 715.

1 H-NMR spectrum (CDCl ₃ , δ, ppm): 0.82 (t, 12H, CH ₃ ), 1.22 (s, 104H, CH ₂ ), 1.41 (s, 18H, C (CH ₃ ) ₃ , 1.58 (s, 8H, βCH ₂ ), 1.95-2.3 (m, 4H, CH ₂ -S), 2.4 (q, 4H, CH ₂ ), 2.9 ( m, 4H, CH ₂ OCO), 4.02 (t, 8H, αCH ₂ ), 4.57 (q, 2H, CH), 4.81 (t, 2H, CH), 5.5 (d, 2H NH-Boc) 7.7 (d, 2H, NH).

Mass spectrum: [M +] 1596.3.

Similarly, 1,1’-bis - [(1,4-dioleyl) -L-glutamyl] -N, N’-bis- (tert-butyloxycarbonyl) cystine is obtained.

Yield 61%.

IR spectrum: (in the film, ν _max , cm ^-1 ): 2920 (СН ₃ ), 2835 (СН ₃ ), 1740 (С = 0), 1640 (С = С), 1680 (1st amide. P .), 1515 (2nd amide p.), 1465 (CH ₂ ), 1370 (CH ₃ ), 1170 (C-O), 840, 765, 715.

1 H-NMR spectrum (CDCl ₃ , δ, ppm): 0.85 (t, 12H, CH ₃ ), 1.25 (s, 80H, CH), 1.43 (s, 18H, C (CH ₃ ) ₃ , 1.60 (s, 8H, βCH ₂ ), 1.8 (m, 16H, CH ₂ -CH =) 1.95-2.25 (m, 4H, CH ₂ -S), 2.35 (q, 4H, CH ₂ ), 2.75 (m, 4H, CH ₂ OCO), 4.05 (t, 8H, αCH ₂ ), 4.57 (q, 2H, CH), 5.5 (d , 2H, NH-Boc), 5.32 (t, 8H, CH =), 8.0 (d, 2H, NH).

Mass spectrum: [M +] 1492.2.

0.3 g (0.2 mmol) 1,1-bis - [(1,4-dihexadecyl) -L-glutamyl] -N, N-bis- (tert-butyloxycarbonyl) cystine is dissolved in 1 ml of anhydrous carbon tetrachloride and treated with 1 ml of trifluoroacetic acid. After 30 minutes, the solvent was removed in vacuo, the residue was dissolved in 40 ml of anhydrous ethyl acetate, and 0.08 ml of triethylamine was added to pH 7-8. The solution was washed with water to pH 7, dried, and the solvent was removed in vacuo.

Obtain 0.24 g (92%) of 1,1'-bis - [(1,4-dihexadecyl) -L-glutamyl] cystine, so pl. 45 ° C.

1 H-NMR spectrum (CDCl ₃ , δ, ppm): 0.82 (t, 12H, CH ₃ ), 1.22 (s, 104H, CH2), 1.58 (s, 8H, βCH ₂ ), 1.95-2.3 (m, 4H, CH ₂ -S), 2.4 (q, 4H, CH ₂ ), 2.9 (m, 4H, CH ₂ OCO), 4.02 (t, 8H , αCH ₂ ), 4.57 (q, 2H, CH), 4.81 (t, 2H, CH), 7.7 (d, 2H, NH).

Mass spectrum: [M +] 1396.1.

Similarly, 1,1'-bis - [(1,4-dioleyl) -L-glutamyl] cystine is obtained.

Yield 87%.

1 H-NMR spectrum (COCO ₃ , δ, ppm): 0.85 (t, 12H, CH ₃ ), 1.25 (s, 80H, CH ₂ ), 1.60 (s, 8H, βCH ₂ ) 1.8 (m, 16H, CH ₂ -CH =) 1.95-2.25 (m, 4H, CH ₂ -S), 2.35 (q, 4H, CH ₂ ), 2.75 (m 4H, CH ₂ OCO), 4.05 (t, 8H, αCH ₂ ), 4.57 (q, 2H, CH), 5.32 (t, 8H, CH =), 8.0 (d, 4H , NH).

Mass spectrum: [M +] 1292.0.

When cooling to 0 ° C, 0.1 ml (0.94 mmol) of formalin, 0.08 ml (1.9 mmol) of 85% formic acid, 0.25 g (0.19 mmol) of 1.1 are placed in a round-bottom flask '-bis - [(1,4-dihexadecyl) -L-glutamyl] cystine and heated at 70 ° C for 12 hours. Then the reaction mass was diluted with 30 ml of chloroform, washed with 50 ml of 0.1N NaHCO ₃ solution and with water until pH 7. The chloroform layer is dried and evaporated in vacuo. Obtain 0.14 g (52%) 1,1'-bis - [(1,4-dihexadecyl) -L-glutamyl] -N, N, bis- (dimethyl) cystine, mp 32-34 ° C.

1 H-NMR spectrum (CDCl ₃ , δ, ppm): 0.82 (t, 12H, CH ₃ ), 1.22 (s, 104H, CH ₂ ), 1.58 (s, 8H, βCH ₂ ) 1.95-2.3 (m, 4H, CH ₂ -S), 2.35 (s, 12 N, CH ₃ ), 2.4 (q, 4H, CH ₂ ), 2.9 (m, 4H, CH ₂ OCO), 4.02 (t, 8H, αCH ₂ ), 4.57 (q, 2H, CH), 4.81 (t, 2H, CH), 7.7 (d, 2H, NH )

Mass spectrum: [M +] 1452.1.

Similarly, 1,1'-bis - [(1,4-dioleyl) -L-glutamyl] -N, N, '-bis- (dimethyl) cystine is obtained.

Yield 51%.

1 H-NMR spectrum (CDCl ₃ , δ, ppm): 0.85 (t, 12H, CH ₃ ), 1.25 (s, 80H, CH ₂ ), 1.60 (s, 8H, βCH ₂ ) , 1.8 (m, 16H, CH ₂ -CH =) 1.95-2.2 (m, 4H, CH ₂ -S), 2.3 (s, 12H, CH ₃ ), 2.35 (q 4H, CH ₂ ), 2.75 (m, 4H, CH ₂ OCO), 4.05 (t, 8H, αCH ₂ ), 4.57 (q, 2H, CH), 5.32 (t, 8H , CH =), 8.0 (d, 4H, NH).

Mass spectrum: [M +] 1348.0.

To a solution of 0.1 g (0.071 mmol) 1,1'-bis - [(1,4-dihexadecyl) -L-glutamyl] -N, N'-bis- (dimethyl) cystine in 1 ml of dry acetonitrile is added 0, 08 ml of methyl iodide and kept in the dark for 12 hours at room temperature. Next, the reaction mass is evaporated and 1 ml of ether is added. The precipitate formed is filtered off. 0.11 g (95%) of 1,1′-bis - [(1,4-dihexadecyl) -L-glutamyl] -N, N’-bis- (trimethyl) cystine diiodide, mp. 73-76 ° C.

1 H-NMR spectrum (CDCl ₃ , δ, ppm): 0.82 (t, 12H, CH ₃ ), 1.22 (s, 104H, CH ₂ ), 1.58 (s, 8H, βCH ₃ ) 1.95-2.3 (m, 4H, CH ₂ -S), 2.45 (t, 4H, CH ₂ OCO), 2.9 (m, 4H, CH ₂ ), 3.6 (s, 18H, C (CH ₃ ) ₃ , 4.02 (t, 8H, αCH ₂ ), 4.57 (q, 2H, CH), 4.81 (m, 2H, CH), 7.7 (d, 2H , NH).

Mass spectrum: [M +] 1482.1.

Similarly, 1,1’-bis - [(1,4-dioleyl) -L-glutamyl] -N, N’-bis- (trimethyl) cystine diiodide is obtained.

Yield 90%.

1 H-NMR spectrum (CDCl ₃ , δ, ppm): 0.85 (t, 12H, CH ₃ ), 1.25 (s, 80H, CH ₂ ), 1.60 (s, 8H, βCH ₂ ) 1.8 (m, 16H, CH ₂ -CH =) 1.95-2.2 (m, 4H, CH ₂ -S), 2.35 (t, 4H, CH ₂ OCO), 2.75 ( m, 4H, CH ₂ ), 3.55 (s, 18H, C (CH ₃ ) ₃ ), 4.05 (t, 8H, αCH ₂ ), 4.57 (q, 2H, CH), 5.32 (t, 8H, CH =), 8.0 (d, 4H, NH).

Mass spectrum: [M +] 1378.0.

Example 6. The toxicity of the compounds of General formula I.

The toxicity of the synthesized compounds of the general formula I in vitro was studied in relation to the cells of the rat's Gasser neuroma. Cationic dimeric amphiphiles are low toxic, because the number of living cells in the system one day after the introduction of lipid dispersions was more than 90% of the initial level.

Example 7. Transfection activity of compounds of General formula I.

The in vitro gene transfer to tumor transformed SCOV-3 cells of the plasmid pEGFP-Nl with the green fluorescent protein gene was studied. High transfection efficiency with cationic dimeric amphiphiles has been shown to be comparable with that for the commercial Lipofectin transfection agent. In vivo transfection with plasmid (pNT β-gal) and liposome dication / phosphatidylcholine (1: 1) transfection was studied in case of intranasal and intravenous (vena porta) injections in mice. Expression of the β-galactosidase gene was detected histochemically by intravenous administration in the spleen. The results obtained are important for the creation of genosomes used in gene transfer protocols for gene therapy of monogenic and oncological diseases.

Claims (2)

1. Cationic dimeric amphiphiles of the general formula I

where R ₁ and R _{2 are the} same or may differ from each other and mean a saturated or unsaturated hydrocarbon chain C _12-22 ; R ₃ and R _{4 are the} same or may differ from each other and mean lower alkyl; R ₅ may have the following meanings: saturated C _3-22 chain; the residue of mono-, di- or triethylene glycol; cystine residue; n and m are the same or may differ from each other and mean an integer from 1 to 3; X ’and Y’ are the same or may differ from each other and mean non-toxic anions, as transfection agents. 2. A method of producing cationic dimeric amphiphiles of the general formula I, where R ₁ and R _{2 are the} same or may differ from each other and mean a saturated or unsaturated hydrocarbon chain C _12-22 ; R ₃ and R _{4 are the} same or may differ from each other and mean lower alkyl; R ₅ may have the following meanings: saturated C _3-22 chain; the residue of mono-, di- or triethylene glycol; cystine residue; n and m are the same or may differ from each other and mean an integer from 1 to 3; X'and Y 'are the same or may differ from each other and mean non-toxic anions, namely, that they carry out the alkylation of the free amino group of diesters of amino acids to a tertiary amino group by reaction with formalin in the presence of 80-85% formic acid and the binding of two molecules of diesters using active derivative of the corresponding spacer in a polar aprotic organic solvent.