MXPA97010078A - New cationic and polycarriatic anifyls; reagents containing them and their - Google Patents

Abstract

The invention relates to novel cationic or polycationic amphiphiles which are capable of forming aggregates with macromolecules, in particular with DNA or RNA, and their distribution is related to prokaryotic or eukaryotic cells. Compounds with the structure of spermyl-dioleoyloxypropyl have proven to be particularly preferred.

Description

NEW CATIONIC AND POLYIC IONIC ANTIFIFILS; REAGENTS CONTAINING THE SAME AND THEIR USE The invention relates to novel cationic and polycationic amphiphiles which are capable of forming aggregates with macromolecules, in particular DNA or RNA, and refers to their distribution in prokaryotic or eukaryotic cells. Amphiphiles (for example surfactants, detergents) have for some time played a general role in daily life. In contrast, only recently has greater attention been paid to cationic amphiphiles through Felgner's pioneering work since they can be used to distribute DNA and RNA in cells and transfect them in this way (PL Felgner et al., Proc. Nati. Acad. Sci., USA 84, 7413-7417 (1987), P. Hawley-Nelson, WO 94/05624 and co-workers, JP Behr, EP 0394111). The individual reagents used for these are very varied, this is mainly due to the fact that they have been found empirically. In this way, it has not been possible to specifically provide a compound for the application in question. Also, the fact that REF: 26530 compounds exist that only have an effect in a mixture with other reagents reveal the complete complexity of the transfection. A review of most of the reagents that have been found so far is given (Bioconjugate Chem., 5, 382-389 (1994) and references cited therein). However, in general, most reagents can form liposomes. The mechanism of transfection by cationic amphiphiles is still poorly understood. It seems credible that the liposomes by means of their positive charge form a more positively charged complex with the DNA and this binds itself to the negatively polarized cell membrane (P.L. Felgner, Nature 337, 387-388 (1989)). However, it is not true how penetration of the cell membrane and transport to the cell nucleus takes place. However, apart from the efficiency of the transfection, it is possible to define a series of other requirements for the new reagents: for efficiency, it should be necessary to portray the cells such as by permeabilizing the cell membrane with DMSO, a detergent such as digitonin or by drag. the reagents should not be toxic, if possible especially not at the most effective concentration, and should preferably be biologically degradable - it should be possible to use them equally for all cells that come into consideration should not have a specificity for certain DNA molecules should also be possible to use them in vivo. This means that besides the toxicity, also the compatibility with the serum is of main meaning. This has frequently been the reason for a drastic decrease in the efficiency of transfection.

Therefore, the object of the invention was to provide suitable reagents that also meet these requirements. This object is achieved by certain cationic amphiphiles, lipids which can be used to distribute anionic compounds such as, for example, DNA in cells and in this process surprisingly exhibit improved properties with respect to the requirements mentioned above.

In particular, the compounds of the general formula I serve as amphiphiles. wherein Ri represents a saturated or unsaturated group of C (0) -C? -23 saturated or unsaturated C? _2 At represents a group 0-R2 in which R2 has designated significance for Rx and may be the same as , or different from Ri. A2 represents an NR3X or a residue of N + R3R R5Y ~ in which R3, R4 which are the same to each other or different, represent hydrogen, an alkyl group with 1 to 4 carbon atoms, a group (CH2) n-OH or (CH2) n-NH2, where n = 2-6, R5 which may be the same as, or different from R3 or R4, denotes hydrogen, an alkyl group with 1 to 4 carbon atoms, a (CH2) n-OH , a (CH2) n-halide or a group ((CH2) mNH) 0- (CH2) n-NH2, in which m is an integer from 2 to 6 and can be equal to, or different from, not in where n can be a number from 2 to 6, I an integer from 0 to 4, X can, in addition to the meaning for R5, have the following meaning: an amidically linked amino acid, an amidically linked peptide or polypeptide, a C group ( 0) -CHR6N (R7) 2, C (0) CHR6N + (R7) 3, C (0) -CHR6N + (Rt) R8 or C (0) - R6 can be a residue (CH2) m-NR7R8, (CH2) m- N + (R7) 2R6 or ( CH2) m-N + (R7) 3 and m can be an integer from 1 to 5, R7 represents hydrogen or an alkyl group with 1 to 4 carbon atoms, R8 represents a group (CH2) n-N (R7) 2 or (CH2) n- N + (R7) 3 in which n is a number from 2 to 4 and R7 can have the meaning stated above, and Y is a pharmaceutically acceptable anion, Bx is an NH [C (O) - (CH2) P-NH] residue zZ in which p is a number from 1 to 6 and q is a number since 0 to 2, Z represents an amino acid amidically bound, or an amidically linked peptide or polypeptide, a group C (0) -CHR6N (R7) 2, C (O) -CHR6N + (R7) 3, C (0) -CHR6N + ( R7) 2R8 or C (0) -CHR6NR7R8, and R6 to R8 and m have the meanings mentioned above, and B; can have the meaning indicated for Ax, and the meaning for Ai is only valid with B and that of A2 is only valid with B2.

Preferred are compounds in which the Ri and Ai residues have alkyl residues of 10 to 20 carbon atoms. In addition to these compounds in which A2 has the meaning of NR3X are preferred and in particular those in which R3 denotes hydrogen and X denotes an amidically linked amino acid, a suitable amino acid derivative, a peptide or polypeptide. Suitable Y pharmaceutically acceptable anions are, in particular, halides, monomethyl sulfate, acetate, trifluoroacetate and phosphate. The following compounds according to the invention have proven to be particularly suitable: 2- (6-carboxyespermil) -1,3-dioleoyloxy-propylamide, 2- (N, N, N, N ', N', N '-hexamethylornityl) -1, 3-dioleo i loxi-propi lamida, 1- (6-carboxiespermil) -2, 3-dioleoiloxy-propylamide and / or 2,3-dioleoyloxy-N- (N- (spermyl) -glycyl) -aminopropane, as well as the corresponding derivatives thereof. However, 2- (6-carboxyespermil) -1,3-dimyristoyloxy-propylamide, 2- (1,1,1,5,5,10,10,14,14, 14-decamethyl 1-) have proven to be suitable. 6-carboxyespermil) -1, 3-dioleoyloxy-pripilamide and / or 2- (N, N, N, N ', N', N'-hexamethyl-lysyl) -1,3-dioleoxyloxypropylamide. Similar compounds such as, for example, amine and amidine derivatives of glycerol and propanediols such as for example 2-aminopropane compounds have already been described for other uses, a suitability for biological applications has not been suggested however (DE-A- 2835369; Woltrom et al., Journal of the Am. Soc. (1951) 73 (8), pp. 3553; Chem. Abs., 59 (8), 1963, column 8586, Fig. 6). Additionally, the present application also relates to the production of reagents with a transfection property. In this connection, the new reagents can also be used as a solution in a water miscible solvent or as an aqueous liposome formulation. When evaluating its transfection efficiency, it is pointed out that: in the serum-free medium they usually exhibit better efficiency than other commercially available products, in the serum-free medium they surprisingly allow a wider horizontal part of the DNA reagent / reagent with a similar transformation efficiency, they have surprisingly the same or better efficiency in the medium containing serum, the efficiency can be further increased by a liposome formulation, surprisingly, the compounds prove to be much less toxic than the compounds with an efficiency db similar transfection in a serum-free system The transfection efficiencies of some selected compounds are shown in Figures 1 and 2, compared to known reagents. The compounds according to the invention are synthesized by normal methods for a person in the art (J March: Advanced Organic Chemistry, John Wiley &Sons, 1985 and M. Bodanszky, Principies of Peptide Synthesis, Springer Verlag, 1984). ) and the terminal products are optionally purified by chromatography, in particular by chromatography with ion exchange. The appropriate reaction schemes are shown in Figures 3 to 7. A prerequisite for the production of new transfection reagents was that the respective compounds are cationic and are suitable for the formation of added aggregates that hitherto are above all the reactants loaded that have resulted in a transfection there are examples in which the DNA can be transported in the cell by means of unloaded reagents. Nevertheless, for this they have to be enclosed then in the liposomes during the preparation of which the molecule to be transported (for example, DNA) can be damaged. In general, the efficiency achieved by these complexes has not been very high (compare, for example, A. Cudd, C. Nicolau in Liposome Tech nolgy (Gregoriadis Ed.), (1984) CRC Press Inc. Boca Raton, Fl.; S.F. Aliño, M. Bobadilla, M. Garcia-Sanz, M. Lejarreta, F. Unda, E. Hilario in Biochem. Biophys, Res. Commun. 192, 174 (1993)).

An additional starting point was that the compounds should be able to form liposomes. It is not clear because almost exclusively the compounds that form liposomes are suitable for transfection. The latest findings (H. Gershon, R. Ghirlando, SB Guttman, A. Minsky in Biochemistry 32./7143 (1993) and B. Sternberg, FL Sorgi, L. Huang in FEBS Letters 356, 361 (1994) showed that it facilitates The formation of structures called meatball noodles that are required for transfection has not yet been clarified whether the same applies to all reagents and how this contributes to the passage through the cell membrane. of these considerations which are shown in the diagrams of Figures 3 to 7. In general, various methods according to the state of the art are available to provide suitable reagents such as for example pharmaceutical formulations.The liposome and ethanolic formulations are use in particular, whereby the efficiency of the various types of liposomes may already differ (for the preparation of liposomes, see for example, H.

Schreier in "Pharmazie in unserer Zeit" 1_1, 97 (1982)). However, the compounds according to the invention can not be used only for transfection as liposomes but also in the form of other aggregates. In the case of the liposome as well as in the case of aggregate formation, additional lipid compounds may be present in addition to the compounds according to the invention. For example, compounds of the phospholipid class are suitable for this; other compounds well known to those skilled in the art could also be used. It should also be considered as surprising that liposomes can be formed from the compounds according to the invention of type 3, 6, 12 or 15 (see examples 3, 6, 12 and 15) which are described herein for the first time time. The transfection was carried out in cells HeLa with the plasmid pSV2-CAT as an example (Gorman, C.M. et al., Mol. Cell. Biol. 2, 1044-1051 (1982)). The transfection efficiency is determined by the CAT-ELISA equipment (Boehringer Mannheim GmbH) and is shown in Figures 1 and 2 compared to known reagents. Due to their low toxicity these compounds (alone or in combination with other lipid compounds) can also be used in vivo. The invention is further clarified by the following examples.

Example 1 Synthesis of 2- (N-tert.-butyloxycarbonyl-L-alanyl) -1,3-dihydroxy-propylamide 1 756 mg (4 mmol) of Boc-L-alanine and 600 μl (4.3 mmol) of triethylamine dissolved in 10 ml of Absolute THF were placed in a round bottom flask with a bubble counter and cooled to -10 ° C. Then, 320 μl (4 mmol) of methyl chloroformate was added and stirred for 30 minutes at 0 ° C. After the addition of 456 mg (5 mmol) of 2-amino-1,3-propanediol, sufficient water was added to form a homogeneous solution and stirred for an additional 1 at room temperature. After removal of the THF, 50 ml of ethyl acetate were added. The organic phase was washed with saturated NaHCO 3 solution and saturated NaCl solution and dried over Na 2 SO 4. After removal of the solvent, 332 mg (32%) of a colorless oil were obtained 1H-NMR (CDC13): d = 1.32 (d; 3H; J = 7.1 Hz; CH-CH3); 1.40 (s; 9H; C (CH3) 3); 3.55-3.85 (m; 4H; CH (CH2OH) 2); 3.85-4.0 m; 1 HOUR; NH-CH); 4.0-4.5 (; 3H; NH-CH, 2 x OH); 5.6-5.8 (m; 1H; NH-CH (CH2OH) 2) 7.0-7.35 (; 1H; OOC-NH-CH). 13 C NMR (CDCl 3): d = 18.4 (CH-CH 3); 28.2 (C (CH3) 3), 52.2 (NH-CH-CO); 52.4 (CH (CH2OH) 2); 61.8 (CH (CH2OH) 2); 80.2 (C (CH3); 155.7 (NH-COO); 173.8 (CH-CO-NH).

Example 2 Synthesis of 2- (N-tert-butyloxycarbonyl-L-alanyl) -1,3-dioleoyloxy-propylamide 2 A solution of 598 mg (2.28 mmol) of 1, 1610 g (5.70 mmol) of oleic acid, 1176 g (5.70 mmol) DCC and 28 mg (0.23 mmol) of dimethylaminopyridine in 40 ml of absolute CH2C12 was stirred overnight room temperature in a round bottom flask. Subsequently, it was filtered and the filtrate was rotary evaporated. The crude product was purified by column chromatography (silica gel, CH2Cl2: MeOH = 30: 1). 1,450 g (80%) of a colorless oil was obtained.

XH NMR (CDC13): d = 0.87 (t; 6H; J = 6.6 Hz; 2 x CH2-CH3); 1.15-1.5 (m; 43H; CH-CH3 / 2 x CO- (CH2) 2- (CH2) 4, 2 x (CH2) 6-CH3); 1.43 (s; 9H; C (CH3) 3); 1.5-1.7 (m; 4H; 2 x CO-CH2-CH2); 1.9-2.1 (m; 8H; 2 x CH2-CH = CH-CH2); 2.30 (t, 4H, J = 7.6 Hz, 2 x CO-CH2); 4.0-4.25 (m; 5H; NH-CH; 2 x CH2OOC); 4.35-4.50 (m; 1H; NH-CH); 9.6-5.0 (; 1H; NH-CH-CH2); 5.25-5.45 (m; 4H; 2x CH = CH); 6.5-6.65 (m; lh; OOC-NH-CH). 13 C NMR (CDC13): d = 14.0 (2 x C (18) H3); 22.5 (2 x C (17) H2); 24.7 (2 x C (3) H2); 27.0, 27.1, 28.98, 29.04, 29.2, 29.4, 29.56, 29.62. { 2 x [C (4) H2-C (7) H2, C (12) H2-C (16) H2]}; 28.1 (C (CH3) 3); 31.8 (2 x C (2) H2); 33.87, 33.88. { 2 x [C (8) H2, C (11) H2]}; 47.2 (CH (CH202C-R) 2); 62.4 (CH (CH202C-R) 2); 129.6, 129.9 (2 x CH = CH); 172.4 (CH = CO-NH); 173.4 (CH2-COO).

Example 3 Synthesis of 2-L-alanyl-l, 3-dioleoyloxy-propylamide 3 702 mg (0.89 mmol) of 2 dissolved in 6 ml of absolute CH2C12 were placed in a round bottom flask with a bubble counter and 2 ml (26.12 mmol) of TFA was added. After 30 minutes of stirring at room temperature, the reaction mixture was transferred to a separatory funnel and 50 ml of CH2C12 as well as 28 ml of 1 N NaOH were added. The organic phase was washed with 10 ml of saturated NaHCO3 solution. and dried over Na2SO4. After removal of the solvent, 599 mg (97%) of an almost colorless oil was obtained.

X H NMR (CDCl 3): d = 0.75-0.9 (; 6 H; 2 x CH 2 -CH 3); 1. 1-1.4 (m; 43H; CH-CH3, 2 x CO- (CH2) 2- (CH2) 4, 2 x (CH2) 6-CH3); 1.4-1.7 (m; 6 H; NH2, 2 x CO-CH2-CH2); 1.85-2.1 (; 8 H; 2 x CH2-CH = CH-CH2); 2.30 (t, 4 H; J = 7.5 Hz; 2 xCO-CH2); 3.35-3.55 (m; 1 H; NH-CH); 4.0- 4.25 (m; 4 H; 2x CH2OOC); 4.3-4.5 (m; 1 H; NH-CH); . 2-5.4 (m; 4 H; 2 x CH = CH); 7.5-7.65 (; 1 H; CO-NH-CH).

NMR 13, (CDCl 3): d = 14.0 (2 x C (18) H3); 22.5 (2 x C (17) H2); 24.7 (2 x C (3) H2); 27.0, 27.1, 28.9, 29.0, 29.2, 29.4, 29.5, 29.6. { 2 x [C (4) H2-C (7) H2, C (12) H2-C (16) H2]}; . 31.7 (2 x C (2) H2); 33.9. { 2 x [C (8) H2, C (11) H2]}; 46.7 (CH (CH202C-R) 2); 62.5 (CH (CH202C-R) 2); 129.5, 129.8 (2 x CH = CH); 173.3 (CH-CO-NH); 173.4 (CH2-COO).

Example 4 Synthesis of 2- (N, N'-di-tert-butyloxycarbonyl-L-ornithyl) -1,3-dihydroxypropylamide 4 2. 000 g (6 mmol) of Boc-L-ornithine and 900 μl (6.45 mmol) of triethylamine dissolved in 25 ml of absolute absolute THF were placed in a round bottom flask with a bubble counter and cooled to -10 ° C. . Then 480 μl (6 mmol) of methyl chloroformate was added and stirred for 30 minutes at 0 ° C. After the addition of 684 mg (7.5 mmol) of 2-amino-1,3-propanediol, sufficient water was added to form a homogeneous solution and stirred for an additional 1 hour at room temperature. After removal of the solvent, it was taken up in ethyl acetate, washed with saturated NaCH03 solution and saturated NaCl solution and dried over NaSO4. After removal of the solvent, 1,987 g (82%) of 4 were obtained as colorless crystals.

NMR: H (CDC13): d = 1.42 (s; 9 H; C (CH3) 3); 1.4-1.95 (m; 4 H; CH2-CH2-CH2-NH); 3.0-3.2 (m; 2 H; CH2NH); 3.6-3.8 (m; 4 H; CH (CH2OH) 2); 3.9-4.05 (m; 1 H; NHCH); 4.05-4.6 (; 3 H; NH-CH, 2 x OH); 5.2-5.4 (m; 1 H; NH-CH); 5.8-6.0.5 (m; 1 H; NH-CH); 7.35-7.5 (m; 1 H; NH-CH). 13 C NMR (CDCl 3): d = 26.1 (CH 2 -CH 2 -CH 2 -NH); 28.4 (C (CH3) 3, 28.5 (C (CH3) 3); 30.1 (CH2-CH2-NH); 40.0 (CH2-NH); 52.9 (CH (CH2OH) 2); 54.4 (NH-CH-CO) 61.8 (CH (CH2OH) 2); 79.3 (C (CH3) 3); 80.1 (C (CH3) 3); 156.1 (NH-COO); 156.5 (NH-COO); 173.3 (CH-CO-NH); .

Example 5 Synthesis of 2- (N, N'-di-tert-butyloxycarbonyl-L-ornithyl) -1,3-dioleoxy-propylamide 5.

A solution of 1898 g (4.68 mmol) of 4, 3. 305 g (11.7 mmol) of oleic acid, 2.414 g (11.7 mmol) of DCC and 57 mg (0.47 mmol) of dimethylaminopyridine in 50 ml of absolute CH2C12 were stirred overnight at room temperature in a bottom flask. Subsequently, it was filtered and the filtrate was rotary evaporated. The crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 30: 1). There were obtained 1678 g (38%) of the product as a waxy, colorless solid of melting point of 52-54 ° C.

NMR aH (CDC13): d = 0.75-0.9 (m; 6 H; 2 x CH2-CH3); 1.05-1.5 (m; 40 H; 2 x CO- (CH2) 2. (CH2) 4, 2 x (CH2) 6- CH3); .1.42 (s; 18 H; 2 x C (CH3) 3; 1.45-1.85 (; 8 H; 2 x CH2-CH2-CH2-NH, 2 x CO-CH2-CH2); 1.85-2.1 (m, 8 H; 2 x CH2-CH = CH-CH2); 2.2-2.35 (m; 4 H; 2 x CO-CH2); 3. 0-3.35 (2 m; 2 H; CH2-NH); 4.0-4.25 (; 5 H; NH-CH; 2 x CH2OOC); 4.35-4.5 (m; 1 H; NH-CH); 4.7-4.8 (m; 1 H; NH-CH); 5.1-5.2 (m; 1 H; NH-CH); 5.2-5.4 (; 4 H; 2 x - CH = CH); 6.65-6.8 (m; 1 H; OOC-NH-CH).

NMR I (CDCl 3): d = 14.0 (2 x C (18) H 3); 22.5 (2 x C (17) H2); 24.6 (2 x C (3) H2); 26.2 (CH2-CH2-CH2-NH); 27. 0, 27.1, 28.97, 29.04, 29.2, 29.4, 29.56, 29.61. { 2 x [C (4) H2-C (7) H2, C (12) H2-C (16) H2] and CH2-CH2-NH}; 28. 1, 28.3 (2 x C (CH3) 3); 31.7 (2 x C (2) H2); 33.85, 33.86. { 2 x [C (8) H2, C (11) H2]} . 47.2 (CH (CH202C-R) 2); 62. 3 (CH (CH202C-R) 2); 129.6, 129.8 (2 x CH = CH); 156.3 (NH-COO); 173.30, 173.33 (CH-CO-NH, 2 x CH2-COO).

Example 6 Synthesis of 2-L-ornithyl-l, 3-dioleoyloxy-pripilamide 6 421 mg (0.451 mmol) of 5 dissolved in 3 ml of absolute CH2C12 were placed in a round bottom flask and 1 ml (13.06 mmol) of TFA was added. After stirring for 30 minutes at room temperature, the reaction material was diluted with 200 ml of CH2C12, washed twice with saturated NaHCO3 solution and dried over Na2SO4. After removal of the solvent, 296 mg (89%) of an almost colorless oil were obtained. 1H NMR (CDC13): d = 0.86 (t; 6 H; J = 6.7 Hz; 2 x CH2-CH3); 1.1-1.45 (m; 40 H; 2 x CO- (CH2) 2- (CH2) 4, 2 x (CH2) 6-CH3); 1.45-1.7 (m; 8 H; CH2-CH2-CH2-NH2, 2 x CO- (CH2-CH2); 1.9-2.1 (m; 8 H; 2 x CH2-CH = CH-CH2); 2.30 (t; 4 H; J = 7.5 Hz; 2 x CO-CH2); 2.6-3.1 (m; 4 H; 2 x NH2); 3.15-3.55 (m; 2 H; CH2-NH2); 3.55-3.75 (m; 1 H; NH-CH); 4.0-4.25 (m; 4 H; 2 x CH2-OOC); 4.3-4.5 (m; 1 H; NH-CH); 5.2-5.4 (m; 4 H; x CH = CH); 7.65-7.75 (; 1 H; CO-NH-CH). 13 C NMR (CDC1): d = 14.0 (2 x C (18) H3); 22.5 (2 x C (17) H2); 24.7 (2 x C (3) H2); 26.99, 27.04, 29.1, 29. 3, 29.5, 29.6,. { 2 x [C (4) H2-C (7) H2, C (12) H2-C (16) H2], CH2-CH2-CH2NH}; 31.7, (2 x C (2) H2); 33.9 (2 x [C (8) H2, C (11) H2]); 46.7 (CH (CH202C-R) 2); 54.6 (NH2-CH-CO); 62.5 (CH (CH202C-R) 2); 129.5, 129.8 (2 x CH = CH); 173.31, 173.6 (CH-CO-NH, 2 x-CH2-COO); 174. 7 (CH-CO-NH).

Example 7 Synthesis of N-tert-butyloxycarbonyl-1,3-dihydroxy-propylamine 7 A solution of 1.07 (5 mmol) of Boc20, of 697 μl (5 mol) of triethylamine and 456 mg (5 mmol) of 2-amino-1,3-propanediol in 10 ml of THF / water (1: 1) was added. stirred overnight at room temperature in a round bottom flask. After removal of the THF in a rotary evaporator, 50 ml of ethyl acetate and 10 ml of saturated NaCl solution were added. The organic phase was washed with 10 ml of saturated NaCl solution and dried over Na 2 SO 4.

After removal of the solvent, 589 mg (62%) of 7 were obtained as colorless crystals. Melting point 83-85 ° C.

X H NMR (CDCl 3): d = 1.44 (s; 9H; C (CH 3) 3); 3.4-3.85 (m; 7H; CH (CH2OH) 2); 5.3-5.45 (; 1H; NH-CH). 13 C NMR (CDCl 3): d = 28.4 (C (CH 3) 3); 53.3 (CH (CH2OH) 2); 62.6 (CH (CH2OH) 2); 79.9 (C (CH3) 3); 156.5 (NH-COO).

Example 8 Synthesis of N-tert-butyloxycarbonyl-1,3-dioleoyloxy-propylamine 8 A solution of 478 mg (2.5 mmol) of 7, 1. 695 g (6 mmol) of oleic acid, 1.28 g (6 mmol) of DCC and 12 mg (0.1 mmol) of dimethylaminopyridine in 25 ml of absolute CH2C12 were stirred overnight at room temperature in a round bottom flask. Subsequently, it was filtered and the filtrate was evaporated on a rotary evaporator. The crude product was purified by column chromatography on silica gel (hexane: ethyl acetate = 5: 1). 1482 g (82%) of the product were obtained as a colorless oil.

NMR: H (CDC13) d = 0.86 (t; 6H; J = 6 Hz; 2 x CH2-CH3) 1.1-1.5 (m; 40H; 2 x CO- (CH2) 2- (CH2) 4 2 x (CH2 ) 6-CH3) 1.43 (s; 9H; C (CH3); 1.5-1.7 (; 4H; 2 x CO-CH2-CH2) 1.9-2.1 (m, 8H; 2 x CH2-CH = CH-CH2); 2.30 (t; 4H; J = 7.5 Hz; 2 x CO-CH2); 3.95-4.25 (m; 5H; NH-CH; 2 x CH2OOC); 4.7-4.85 (m; 1H; OOC-NH-CH); 5.25-5.45 (m; 4H; 2 x CH = CH). 13 C NMR (CDClj): d = 14.0 (2 x C (18) H3); 22.5 (2 x C (17) H2); 24.7 (2 x C (3) H2); 27.0, 27.1, 28.96, 29.02, 29.2, 29.4, 29.55, 29.62. { 2x [C (4) H2-C (7) H2, C (12) H2-C (16) H2]}; 28.2- (C (CH3) 3); 31.8 (2 x C (2) H2); 33.9. { 2 x [C (8) H2, C (11) H2]}; 48.4 (CH (CH202C-R) 2); 62.8 (CH (CH202C-R) 2); 79.8 (C (CH3) 3); 129.6, 129.8 (2 x CH = CH); 154.9- (NH-COO); 173.3 (CH2-COO).

Example 9 Synthesis of 1,3-dioleoyloxy-propylamine 9 720 mg (1 mmol) of 8, dissolved in 3 ml of Absolute CH2C12 was placed in a round bottom flask and 1 ml (13.06 mmol) of TFA was added.

After stirring for 30 minutes at room temperature, the reaction material was diluted with 50 ml of CH 2 C 12, washed with saturated NaHCO 3 solution and dried over Na 2 SO 4. After removal of the solvent, 589 mg (95%) of a colorless oil was obtained.

NMR IE (CDC13): d = 0.87 (t; 6H; J = 6.6 Hz; 2 x CH2-CH3); 1.1-1.5 (m; 40H; 2x CO- (CH2) 2- (CH2) 4, 2 x (CH2) 6-CH3); 1.35-1.5 (m; 2H; CH-NH2); 1.5-1.7 (m; 4H; 2 x CO- (CH2-CH2); 1.85-2.1 (m; 8H; 2 x CH2-CH = CH-CH2); 2.32 (t; 4H; J = 7.5 Hz; 2 x CO-CH2), 3.2-3.35 (m; 1H; CH-NH2); 3.95-4.15 (m; 4H; 2 x CH2-OOC); 5.25-5.45 (m; 4H; 2 x CH = CH). 13 C NMR (CDCl 3): d = 14.0 (2 x C (18) H 3); 22.5 (2 x C (17) H2); 24.8 (2 x C (3) H2); 27.0, 27.1, 28.96, 28. 99, 29.03, 29.2, 29.4, 29.55, 29.63 (2 x [C (4) H2-C (7) H2, C (12) H2-C (16) H2].}., 31.8, (2 x C ( 2) H2); 34.0 (2 x [C (8) H2, C (11) H2]); 49 {CH (CH202C-R) 2.};; 65.7 . { CH (CH202C-R) 2}; 129.6, 129.9 (2 x CH = CH); 173.4 (CH2-COO).

EXAMPLE 10 Synthesis of 2- (N, N ', N ", N' '' -tetra-tert-butyloxy-carbonyl-6-carboxy-spermyl) -1,3-dihydroxy-propylamide μl (312 μmol) of methyl chloroformate at -10 ° C was added to a solution of 202 mg (312 μmol) of tetra-Boc-sper ina and 47 μl (335 μmol) of rietila ina in 2 ml of absolute THF. and were stirred for 30 minutes at 0 ° C. After the subsequent addition of 36 mg (390 μmol) of 2-amino-1, 3-orthopanediol, sufficient water was added to form a homogeneous emulsion and then stirred for an additional 1 hour at room temperature. After removal of the solvent in a rotary evaporator, approximately 30 ml of ethyl acetate were added. The organic phase was washed with saturated NaHCO 3 solution and saturated NaCl solution. And dried over Na2So. After removal of the solvent, 208 mg (93%) of a foamy, colorless solid was obtained. 1N NMR (CDC13): d = 1.3-1.47 (; 36H; 4 x C (CH3) 3); 1. 5-2.05 (m; 8H; 2 x N-CH2-CH2-CH2-N, N-CH-CH2-CH2-CH2-N); 2.85-3.4 (; 10H; 5 x N-CH2); 3.65-3.8 (m; 4H; 2 x CH2-OH); 3.8-4.5 (m; 4H; 2 x -CH-, 2 x CH2-OH); 4.8-5.1 (s (broad); 1H; CH2-NH); 5.25-5.4 (s (broad); 1H; CH2-NH); 7.05-7.15 (s (broad); 1H; -NH-CO). 3 C NMR (CDC13): d = 25.0, 29.5 (N-CH-CH2-CH2-CH2-N) 28. 0, 28.2, 28.3 (4 x C (CH3) 3); 38.0, 38.1 (2 x N-CH2-CH2-CH2-N); 42.9, 45.0, 47.0 (other spermyl-CH2 carbon); 52.4 (CH (CH2-OH) 2); 59.8 (CH-CO); 63.1 (CH (CH2-OH) 2); 79.3, 79.5, 81.1 (4 x CO-O-C) CH3) 3) 156.0, 156.1 (4 x CO-O-C) CH3) 3); 171.1 (CO-NH).

EXAMPLE 11 Synthesis of 2- (N, N ', N ", N' '' -tetra-tert-butyloxycarbonyl-6-carboxy-aperol) -1,3-dioleoyloxy-propylamide A solution of 208 mg (72 μmol) 10, 204 mg (289 μmol) of oleic acid, 149 mg (723 μmol) of DCC and 4 mg (29 μmol) of dimethylaminopyridine in 6 ml of absolute CH2C12 was stirred overnight ambient temperature in a closed round bottom flask. Subsequently, it was filtered, washed again with hexane / ether (3: 1) and the filtrate was evaporated on a rotary evaporator. After column chromatography on silica gel (CH2Cl2 / MeOH = 20: 1). 160 mg (44%) of a colorless oil was obtained.

X H NMR (CDCl 3): d = 0.8-0.95 (m; 6H; 2 x CH 2 -CH 3); 1.2- 1.4 (m; 40H; 2 x CO- (CH2) 2- (CH2) 4, 2 x (CH2) 6-CH3); 1.4-1.55 (m; 36H; 4 x C (CH3) 3); 1.55-1.75 (m; 10H; 3 x (-CH2-CH2-CH2-) spßrm 2 x CO-CH2-CH2-); 1.85-2.05 (m, 10H; (-CH-CH2-CH2-) 3pßtm, 2 x CH2-CH = CH-CH2); 2.25-2.35 (m; 4H; 2 x CO-CH2); 3.0-3.35 (m, 10H; 5 x N-CH2); 4.0-4.4 (m; 6H; 2 x -CH-N, (CH (CH202C-R) 2); 4.5-5.1 (s (very broad); 1H; NH-CH2); 5.25-5.35 (; 4H; 2 x CH = CH); 6.8-7.0 (s (broad); 1H; NH-CO). 13 C NMR (CDC13): d = 14.4 (2 x C (18) H3); 23.0 (2 x C (17) H2); 5.2 (2 x C (3) H2); 27.6 (C (8) H2 C (11) H2); 29.5, 29.6, 29.7, 29.9, 30.1, 32.3 ((CH-CH2-CH2-CH2.) Sperm, 2 x N- CH2-CH2 -CH2 ~ N, 2 XC (4) H2 ~ C (7) H2, 2 XC (12) H2-C (16) H2; 28.7, 28.8 (4 x C (CH3) 3); 34.0 (2 x C (2) H2); 38.5, 44.6, 46.8, 47.6 (2 x N-CH2- CH2-CH2-N- (CH-CH2-CH2-CH2-) spe m (CH (CH202C-R) 2); 59.5 (CO-CH); 62.8 (CH (CH202C-R) 2); 79.5, 80.1, 81.5 (4 x CO-OC (CH3) 3), 130.07, 130.37 (2 x CH = CH), 156.3 (4 x CO-OC (CH3) 3), 173.7 (CO-NH, 2 x CH2-COO).

Example 12 Synthesis of 2- (6-caarboxy-spermyl) -1,3-dioleoyloxy-propylamide 12 85 mg (68 μmol) of 11 were dissolved in 1 ml of absolute CH2C12 in a round bottom flask with bubble counter and 400 μl of TFA was added. The reaction mixture was stirred for 45 minutes at room temperature. Subsequently, approximately 20 ml of CH2C12 were added, washed with saturated NaHCO3 solution and dried over Na2So. After removal of the solvent, 56 mg (97%) of a colorless oil was obtained.

AH NMR (CDCl3): d = 0.8-0.95 (m; 6H; 2 x CH2-CH3); 1. 15-1.4 (m; 40H; 2 x CO- (CH2) 2- (CH2) 4, 2 x (CH2) 6-CH3); 1.5-1.9 (m; 12H; 3 x (-CH2-CH2-CH2-) 3pßrp ?, 2 x CO-CH2-CH2-, (-CH-CH2-CH2-) sp? Rm); 1.9-2.1 (m, 8H; 2x CH2-CH = CH-CH2); 2.2-2.35 (m; 4H; 2 x CO-CH2); 2.45-3.3 (; 10H; 5 x N CH2); 4.0-4.59 (m; 12H; 2 x CH-N, (CH (CH202C-R) 2, 2 x NH2, 2 x NHspßrm) / 5.25-5.45 (m; 4H; 2 x CH = CH); 7.6-7.9 (s (broad); 1H; NH-CO). 13 C NMR (CDCl 3): d = 14.0 (2 x C (18) H 3); 22.5 (2 x C (17) H2); 24.7 (2 x C (3) H2); 27.0, 27.1 (C (8) H2, C (11) H2); 29.0, 29.1, 29.2, 29.4, 29.60, 29.61, 31.8 - ((CH-CH2-CH2-CH2-) Sperm, 2 x N ~ CH2 -CH2-CH2 ~ N, 2 x C (4) H2-C (7 ) H2, 2 x C (12) H2-C (16) H2); 33.9 (2 x C (2) H2); 41.0, 47.3, 48.8 (2 x N-CH2-CH2-CH2-N, (CH-CH2-CH2-CH2-) sp? Rm, (CH (CH202C-R) 2); 62.0, 62.9 (CO-CH, CH (CH202C-R) 2); 129. 5, 129.9 (2 x CH = CH); 173.4, 174.2 (CO-NH, 2 x CH2-COO).

Example 13 Synthesis of 2-dimethylamino-1,3-propanediol 13 456 mg (5 mmol) of 2-amino-1,3-propanediol were placed in a round bottom flask and mixed with 1.13 ml (25 mmol) of formic acid (85%, d = 1.20) while cooling ice. Subsequently, 895 μl (12 mmol) of formaldehyde (37%) were added and the mixture was heated for 10 hours at 80 ° C in a water bath. After cooling 3 ml of 2N hydrochloric acid were added. The product was isolated by chromatography on a 25 g acid ion exchanger (DOWEX). The crude product obtained in this way was purified by short path distillation in an oil pump vacuum. 253 mg (42%) of a colorless oil was obtained.

Boiling point? Mbar = 105 ° C.

NMR? K (DMSO-dg); or = 2.25 (s; 6H; N (CH3) 2); 2.31-2.45 (, 1H; CH (CH2OH) 2); 3.35-3.55 (m, 4H; CH (CH2OH) 2); 4.24 (s (broad); 2H; CH (CH2OH) 2). 13 C NMR (DM80-d6); d = 41.8 (N (C_H3) 2); 58.7- (CH (CH2OH) 2); 66.7 (CH (CH2OH) 2).

Example 14 Synthesis of 2-dimethylamino- (1,3-dioleoyloxy) -propane 14 A solution of 183 mg (1.54 mmol) of 13, 1.088 g (3.85 mmol) of oleic acid, 795 mg (3.85 mmol) of DCC and 18 mg (154 μmol) of dimethylamine pyridine in 20 ml of absolute CH2C12 was stirred overnight at room temperature.

Subsequently, it was filtered, washed again with hexane / ether (3: 1) and the filtrate was evaporated on a rotary evaporator. After column chromatography on silica gel, (CH2Cl2 / MeOH = 30: 1) 444 mg (45%) of the product was obtained as a colorless oil.

XH NMR (CDCl3): d = 0.85-0.95 (m; 6H; 2 x CH2-CH3) 1.2-1.4 (m; 40H; 2 x CO- (CH2) 2- (CH2) 4, 2 x (CH2) 6 -CH3) 1.55-1.7 (m; 4H; 2 x CO-CH2-CH2-); 1.95-2.05 (m; 8H 2 x CH2-CH = CH-CH2); 2.3-2.35 (, 4H; 2 x CO-CH2-CH2-) 2.39 (s; 6H; N (CH3) 2); 2.9-3.0 (m; 1H; CH (CH202C-R) 2) 4.1-4.3 (m; 4H; CH (CH202C-R) 2); 5.3-5.4 (m; 4H; 2 x CH = CH).

AH NMR (CDCl 3): d = 14.4 (2 x C (18) H 3); 23.0 (2 x C (17) H2); 25.3 (2 x C (3) H2); 27.55, 27.6 (C (8) H2; C (11) H2); 29.50, 29.53, 29.67, 29.7, 29.8, 29.9, . 06, 30.14, 32.3 (2 x C (4) H2-C (7) H2, 2 x C (12) H2-C (16) H2); 34.7 (2 x C (2) H2); 42.4 (N (CH3) 2); 61.6, 61.9 (CH (CH202C-R) 2); 130.09, 130.36 (2 x-CH = CH); 173.9 (CH2-COO) Example 15 Synthesis of N- [2- (1, 3-dioleoyloxy) propyl] -N, NN-trimethylamoniorethyl sulfate 15 66 μl (696 μmol) of dimethyl sulfate at 0 ° C was added to a solution of 200 mg (309 μmol) of 14 in 1.55 ml of ethyl acetate / hexane (1: 1) and the reaction material was filtered through 24 hours at 4 ° C. Subsequently, the solvent was removed. 213 mg (89%) of a colorless oil were obtained.

XH NMR (CDC13): d = 0.75-0.9 (m; 6H; 2 x CH2-CH3); 1.05-1.4 (m; 40H; 2 x CO- (CH2) 2- (CH2), 2 x (CH2) 6-CH3); 1.4-1.65 (; 4H; 2 x CO-CH2-CH2-); 1.8-2.1 (m; 8H; 2 x CH2-CH = CH-CH2); 2.25-2.4 (, 4H; 2 x CO-CH2- CH2-); 3.3-3.45 (s (broad); 9H; N (CH3) 3); 3.61 (s; 3H; CH3-0-S); 4.1-4.2 (m; 1H; CH (CH202C-R) 2); 4.4-4.65 (m; 4H; CH (CH202C-R) 2); 5.2-5.4 (m; 4H; 2 x CH = CH).

NMR 13, (CDCl 3) d = 13.8 (2 x C (18) H 3); 22.4 (2 C (17) H2); 24.4 (2 x C (3) H2); 26.9, 27.0 (C (8) H2; C (11) H2); 28.8, 28.9, 29.0, 29.05, 29.3, 29.46, 29.5, 31.6 (2xC (4) H2-C (7) H2, 2 x C (12) H2-C (16) H2); 33.6 (2 x C (2) H2); 53.0, 54.0 (N (CH3) 3, CH3-0-S); 58.7- (CH (CH202C-R) 2); 69.8 (CH (CH202C-R) 2); 129.4, 129.8 (2 x CH = CH); 172.3 (CH2-C_00).

Example 16 Synthesis of N, N ', N ", N' '' - tetra-tert-butyloxycarboni 1-6-carboxy-spermi 1-2, 3-dihydroxy-l-propylamide 16 A solution of 300 mg (0.47 mmol) of tetra-Boc-carboxyspermine in 4 ml of dichloromethane was mixed successively with 59 mg (0.51 mmol) of hydroxysuccinimide dissolved in 2 ml of dichloromethane / THF (1: 1) and 106 mg (0.51 mmol). mmol) of dicyclohexylcarbodiimide dissolved in 2.2 ml of dichloromethane and stirred for 4 days at room temperature. After removal of the solvent, the residue was taken up in ethyl acetate, filtered and the solvent was removed from the clear filtrate. The residue (335 mg (0.45 mmol) tetra-Boc-carboxypermine-hydroxy-succinimidyl ester) was taken in 2.18 ml of dimethylformamide and mixed with 41 mg (0.45 mmol) of (±) -l-amino-2, 3 -propanediol and 61.5 μl (0.45 mmol) of triethylamine. After stirring for 3 days, the solvent was removed and the residue was diluted between ether and water. The organic phase was dried. After removal of the solvent, an oily product was obtained and processed directly.

Example 17 Synthesis of N, N ', N ", N' '' -tetra-tert-butyloxycarbonyl-6-carboxy-spermyl-2,3-dioleoyloxy-1-pripylamide 17 218 mg (0.77 mmol) of oleic acid, 180 mg (0.87 mmol) of dicyclohexylcarbodiimide and a catalytic amount of dimethylaminopyridine were added to a solution of 252 mg (0.35 mmol) of 16 in 1.4 ml of carbon tetrachloride. It stirred during the night. After removal of the solvent, the residue was chromatograp(silica gel, dichloromethane with an increasing methanol content up to 10%). 392 mg (92% of theory) of an oil were obtained. 1E NMR: d = 173.8, 173.3 (oleic acid-C = 0), 156 (C (= 0) -N, 130.0, 129.7 (oleic acid C = C), 80.97, 79.67, 79.84 (quat.-BOC), 70.3 (Glic.sn2), 62.6 (Glic.snl), 59.1 (sperm.-), 46.5, 44.3, 43.7, 43.1 (sperm.N-CH2), 39.5 (Glic.sn3), 38.2 (sperm -CH2) , 34.9 (oleic acid-C-2), 31.9 (oleic acid C-16), 29.8-29.1 (oleic acid-CH2), 28.4 (BOC-CH3), 27.2 (oleic acid C-8, 11), 25.0 ( oleic acid C-3), 22.7 (oleic acid C-17), 14.1 (oleic acid C-18).

Example 18 (6-carboxy-spermyl) -2, 3-dioleoyloxy-1-propylamine 18 A solution of 195 mg (0.16 mmol) of 17 in 5 ml of dichloromethane was stirred at room temperature with 5 ml of TFA. After stirring for 20 minutes, it was diluted with 100 ml of dichloromethane and waswith saturated sodium hydrogen carbonate solution. The combined organic phases were dried and the solvent was evaporated. 134 mg (99%) of a viscous oil was obtained.

NMR XH (500 MHz, CDCl3 / CD3COOD 5: 1) d = 5.33 (oleic acid 9, 10-H), 5.16 (glycerol 2-H), 4.25 and 4.06 (glycerol 1-H), 4.15 (spindle-H), 3.55 and 3.33 (glycerol 13-H), 3.18 and 3.05 (spermine N-CH2), 2.31 (oleic acid 2-H), 2.21 (spermine-CH2), 2.01 (oleic acid 8, 11-H), 1.98 (spermine-), 1.83 (spermine CH2), 1.57 (oleic acid 3-H), 1.30 (oleic acid 4-7, 12-17-H), 0.87 ppm (oleic acid 18-H) 13 C NMR: d = 173.82 and 173.53 (oleic acid Cl), 154.06 (C (= 0) -N), 130.17 and 129.81 (oleic acid C-9 and C-10), 70 (glycerol C-2), 63.2 ( glycerol Cl), 39.8 (glycerol C-3), 34.25, 34.05, 33.97 (oleic acid C-2), 32.70, 31.93 (oleic acid C-16), 30.82, 29.79, 29.57 and 29.35 (oleic acid CH2), 27.25 (oleic acid C-8, 11), 25.64, 25.53, 25.32, 24.97 (oleic acid C-3), 24.71, 22.70 (oleic acid C-17), 14.13 ppm (oleic acid C-18). MS (FAB): MH + 848.6 (cale.848.8).

Example 19 Synthesis of N- ((N-Boc) -glycyl) -aminopropane-2,3-diol 19 .00 g (36.73 mmol) of Boc-Gli-Osu (Bachem) together with 3.35 g (36.77 mmol) of l-amino-2, 3-propanediol in 50 ml of DMF and stirred for 18 h. The product was purified by flash chromatography (CH2Cl2 / MeOH: 95: 5 v / v).

Obtained 8.94 g (98%) of 19 as a colorless oil.

NMR aH (CDC13): d = 1.45 (s; 9H; CH3); 3.28 (m; 1H; CH2-0H; 3.39 (m; 1H; CH2-OH;); 3.35 (m; 1H; CH2-NaminoProP /); 3.38 (m; 1H; CH2-Namin? Pr? P.); 3.78 (s; 3H; -CH-OH; CH2-NHgli); 4.41 (broad; 1H; OH); 4.67 (broad; 1H; OH); 6.04 (tr; 1H; NH); 7.45, (tr.lH; NH). 13 C NMR (CDC13): (= 28.4 (CH3); 42.2 (CH2-Ngli) '; 44.0 (CH2-Naminoprop /); 64.0 (CH2-OH); 70.8 (CH-OH); 80.3 (quat.CBoc); 156.6 (NC02); 174.5 (NCO).

Example 20 Synthesis of 2,3-dioleoxyloxy-N- ((N-Boc) -glycyl) -aminopropane 8.90 g (35.85 mol) of 19 were dissolved together with 22.70 g (110.02 mmol) of DCC and 26.60 g (94.17 mmol) of oleic acid in 50 ml of DMF. 0.42 g (3.44 mmol) of DMAP was added and stirred for 2 d. After it was filtered, it was evaporated on a rotary evaporator and the product was purified by chromatography (CH2Cl2 / MeOH: 99: 1 v / v). Yield 19.25 g of 20, as a colorless oil (60%).

X H NMR (CDCl 3): (= 0.89, (tr.6H; CH 3, oleic acid); 1. 30 (broad; 40H; C-CH2-C); 1.45 (broad; 9H; CH3'BOC); 1.62 (m; 4H; CH2-CH2C = 0); 2.03 (m; 8H; CH2- CH = CH); 2.32 (tr; 4H; CH2C = 0); 3.42-3.57 (m; 2H; CH2-Namlr.0pr0p.); 3.75 (d; 2H; CH2-Ngli); 4.12 (dd; 1H; CH2-0aminopl: Op.); 4.27 (dd; 1H; CH2-Oaminoprop.); 5.08 (; 1H; CH-O); 5.20 (s, 1H; NH); 5.34 (m; 4H; CH = CH); 6.58, (tr.1H; NH). 13 C NMR (CDC13): (= 14.0 (CH3, oieic acid); 22.7 (CH2, olic acid) 24.9 (CH2, olic acid) / 27.2 (CH2, olic acid) / 27. 2 (CH2, oic acid) 28.3 (CH2, oiesic acid) / 29.2 (CH2, oiesic acid) / 29.3 (CH3, BOC); 29.5 (CH2, acidic acid) 29.8 (CH2, olic acid) / 31.9 (CH2, olic acid) / 34.0 (CH2, oic acid); 34.2 (CH2, olic acid); 39.7 (CH2-Ngii); 44.5 (CH2 - Namin0prop >); 62.7 (CH2_Oaminoprop.); 70.2 (CH - Oaminoprop. ); 80.3 (quat.CBOC); 129.7 (CH = CH); 129.7 (CH = CH); 130.0 (CH = CH); 130.0 (CH = CH); 156.1 (NC02); 169.8 (NCO); 173.2 (Oleic acid); 173.4 (Oleic acid).

Example 21 Synthesis of 2,3-dioleoyloxy-N- (glycyl) -aminopropane 21 .20 g (19.56 mmol) of 20 in 200 ml of CH2C12 / TF (3: 1 v / v) were taken and stirred for 30 min. Then the solution was diluted with 200 ml of CH2C12 and stirred with 200 ml of saturated NaHCO3 solution. The organic phase was dried over MgSO 4 and evaporated on a rotary evaporator. Yield: 11.64 g (88%) of 21 as an oil.

NMR L (CDCl 3): (= 0.88 (tr.6H; CH3); 1.29 (broad; 40H; C-CH2-C); 1.62 (s; 4H; CH2-CH2C = 0); 1.82 (s; 2H CH2- Ngly), 2.01 (m; 8H; CH2-CH = CH); 2.32, (tr.tr 4H; CH2C = 0); 3.36 (s; 2H; NH2); 3.43-3.62 (m; 2H CH2-Naminoprop.); 4.12 (dd; 1H; CH2-Oaminoprop.) 4.28 (dd; 1H; CH2-Oaminoprop.); 5.14 (m; 1H; CH-Oaminoprop.); 5.34 (m; 4H; CH = CH); 7.60, ( tr.lH; NH) 13 C NMR (CDCl 3): (= 14.0 (CH3); 22.6 (CH2, oleic acid); 24.8 (CH2, oleic acid); 24.8 (CH2, oleic acid); 27.1 (CH2, oleic acid) ), 29.0 (CH2, oleic acid), 29.1 (CH2, oleic acid), 29.2 (CH2, oleic acid), 29.4 (CH2, oleic acid), 29.6 (CH2, oleic acid), 31.8 (CH2, oleic acid); 34.0 (CH2, oleic acid), 34.2 (CH2, oleic acid), 39.1 (CH2-Ngly) 44.5 (CH2-Naminoprop.); 62.7 (CH2-Naminoprop.) 129.6 (oleic acid); 129.9 (oleic acid) 173.1 ( NCO), 173.1 (oleic acid), 173.3 (oleic acid).

EXAMPLE 22 Synthesis of 2,3-dioleoyloxy-N- (N- (N, N '-N "-N' '' -tetra-tert-butyloxycarbonyl-6-carboxy-spermyl) -glycyl) -aminopropane 100 mg (0.15 mmol) of 21 were dissolved together with 102 mg (0.16 mmol) of (Boc) -spermil-COOH and 39 mg (0.19 mmol) of DCC in 1 ml of CH2C12 and stirred for 18 h. The precipitated DCH was then removed by filtration. Concentration by evaporation and chromatographic purification (CH2Cl2 / MeOH, 97: 3) yielded 123.1 mg (65%) of 22 as a colorless oil.

NMR aH (CDC13 + 0.11 ml CD3COOD): (= 0.88 (tr; 6H; CH3, oleic acid); 1.28 (broad; 40H; C-CH2-oleic acid); 1.46 (broad; CH3, BOC; CH2, expected); 1.62 (m; 4H; CH2CH2C = 0); 2.03 (m; 8H; CH2-cH = CH); 2.32 (m; 4H; CH2c = 0); 3.00- 3.33 (m; 10H; CH2, spermil); 3. 33-3.62 (m; 2H; CH2- Naminoprop.); 3.95 (m; 2H; CH2-Ngly); 4.10 (m; 1H; CH2- O); 4.28 (m; 1H; CH2-0); 4. 30 (broad; 1H; CHespermil); 5.12 (m; 1H; CHaminoprop.); 5.35 (m; 4H; CH = CH). 13 C NMR (CDCl 3 + 0.1 ml CD 3 COOD): (= 14.1 (CH 3, oleic acid); 22.8 (CH 2, oleic acid); 25.0 (CH 2, oleic acid); 27.3 (CH 2, oleic acid); 27.3 (CH 2, oleic acid);; 28.4 (CH3, BOC); 28.5 (CH3, BOC); 29.3 (CH2, oleic acid); 29.3 (CH2, oleic acid); (CH2, oleic acid); 29.7 (CH2, oleic acid); 29.9 (CH2, oleic acid); 32.0 (CH2, oleic acid); 34.2 (CH2, oleic acid); 39.7 (CH2-Ngly); 43.1 (CH2- Naminoprop.); 63.0 (CH2- O); 70.2 (CH-O); 79.6 (quat.CBOC); 80.2 (quat.CBOC); 80.9 (quat.CBOC); 81. 5 (quat.CBOC); 129.8 (CH = CH); 130.1 (CH = CH); 155-158 (NC02); 173.6 (COO) '.173.8 (COO).

Example 23 Synthesis of 2,3-dioleoyloxy-N- (N- (6-carboxy-spermyl) -glycyl) -aminopropane 23 1.00 g (0.77 mmol) of 22 was dissolved in 100 mL of CH2C12 / TFA (3: 1) and stirred for 30 minutes. Subsequently the solvent was removed, 1.03 g (98.80%) of 23 was obtained as a waxy product.

XH NMR (d6-DM80): (= 0.85, (tr.6H; CH3, oleic acid); 1.23 (broad; 44H; C-CH2-oleic acid / spermil); 1.50 (m; 4H; CH2- CH2C = 0 ); 1.67 (m; 2H; C-CH2-Cespermil); 1.83 (m; 2H; C-CH2-Cespermil); 1.95 (m; 8H; CH2-CH = CH); 2.23 (m; 4H; CH2c = 0 ); 2.82-3.30 (m; 10H; CH2, spermil); 3.26 (m; 1H; CH2-Na inoprop.); 3.36 (m; 1H; CH2-Naminoprop.); 3.74-3.88 (; 2H; CH2-Ngly ); 3.93 (broad; 1H; CHespermil); 4.01 (m; 1H; CH2-0); 4.24 (m; 1H; CH2-O); 5.02 (m; 1H; CHaminoprop.); 5.31 (m; 4H CH = CH), 8.10 (broad; 6H; NH), 8.33, (tr.lH NHC = 0), 8.98 (broad; 2H; NH), 9.04, (tr.lH NHC = 0) '.9.15 (broad; 1H; NH); 9.50 (broad; 1H NH). 13C NMR (d6-DMSO): (= 13.8 (CH3, oleic acid); 20.9 (CH2, esper); 22.1 (CH2, oleic acid); 23.8; 23.9 (CH2, esper), 24.4, 26.6 (CH2, oleic acid), 26.9 (CH2, esper), 28.5-29.1 (CH2, oleic acid), 31.3 (CH2, oleic acid), 33.4.33.6 (CH2, oleic acid), 36.2 ( CH2, esper); 38.7 (CH2, esper); 41.8 (CH2-Ngly); 43.0 (CH2-Naminoprop.); 43. 9.46.2 (CH2, esper); 58.8 (CHsper); 62.8 (CH2-0); 129.5 (CH = CH); 129.6 (CH = CH); 167.4.168.5 (NCOesper / gly); 172.2 (COO); 172.4 (COO).

Example 24 Transfection of adherent HELA cells with CAT plasmid 1. Start Material 1. Medium for the concentrated culture of HELA (ATCC No. CCL 2): MEM (with EARL salts), 10% FCS, 2 mM pivurate, 2 mM glutamine, 1 x n.e. amino acids . For transfection prepare the same medium containing 5% FCS and the same medium without FCS. 2. Plasmid pSV2-CAT in TE buffer, concentration: 1 mg / ml, 5 Kb (Gorman C.M. et al., Mol.Cell. Biol. 2, 1044-1051 (1982)). 3. New transfection reagents: in MES (20 mM MES, 150 mM NaCl, pH 6.2) concentration 1.0 mg / ml, sound treated (Branson sonicator) sterilized by filtration in 99.8% EtOH, 2.5 mg / ml concentration (not treated with sound and not sterilized by filtration) 4. HEPES: 20 mM Hepes, 150 mM NaCl, pH 7.4, sterile, . PBS, BM Order No. 210 331 (10 mM buffer, 150 mM salt), sterile and non-sterile 6. 150 mM NaCl, 7. Shock absorber: 10 mM MOPS, 10-mM NaCl, 1 mM EGTA, 1% mM Triton X-100, pH 6.5 2. Transfection mixture One day before transfection the cells were transferred to 6 cm petri dishes: for this the cells were treated with trypsin and diluted to 2 x 10 5 cells / ml (cell count determined with a Neubauer count chamber) in the medium containing 5% FCS. 5 mi per box. Incubation in an incubator (at 37 ° C, C02 at 5%).

Mixing per box for aqueous solutions of the transfection reagent (TR): 1. Add HEPES to 5 μg of plasmid (- 5 μl) to a final volume of 100 μl and vortex. 2. Add HEPES to 10 - 40 μg of TR (= 10 - 40 μl) to a final volume of 100 μl and vortex. 3. Combine the solutions of 1. and 2., shake carefully. Let stand for 10 to 15 min at Temp. Ambient. 4. During the incubation period change the medium in the test boxes: aspirate the old medium and replace with 3 ml of medium (containing 5% FCS or, if it is without 5% FCS, wash the cells twice with PBS) .

. Add the plasmid-TR mixture from 3 (200 μl) directly to the fresh medium (with or without FCS) and disperse uniformly by careful disk stirring. 6. Incubate for 6 h in an incubator (at 37 ° C, C02 at 5%). 7. Then supplement the medium to a final FCS concentration of 5-10%. Final volume of the medium of 6 mi. 8. Incubate for 19 h in an incubator. 9. Then fully aspirate the mixture and replace with 5 ml of medium containing 5-10 FCS at 5 - 10%.

Mix by disc for TR ethanolic solutions: A. Without FCS in the middle 1. 5 μg of plasmid (= 5 μl) + 500 μl of the medium without FCS, and vortex. -. 10 - 40 μg of RT (= 4 - 16 μl) + 500 μl of medium and wash twice with PBS; Aspire.

Wash the FCS-free cells: aspirate 5 ml of the medium and wash twice with PBS; Aspire.

Combine the solutions of 1. and 2., shake carefully.

Place the plasmid-TR mixture of 4. (approximately 1020 μl) in the washed cells and distribute equally by carefully stirring the box.

Incubate for 6 h in an incubator (at 37%, C02 at 5%).

Then supplement the medium to a final FCS concentration of 5-10 Final volume of the medium of 6 ml.

Incubate for 18 h in an incubator Then fully aspirate the mixture and replace with 5 ml of the medium containing FCS at 5 - 10%.

B. with FCS in the middle 1. 5 μg of plasmid (= 5 μl) + 500 μl of NaCl, and vortex. 2. 10 - 40 μg of TR (= 4 - 16 μl) + 500 μl of NaCl and vortex. 3. Combine the solutions of 1. and 2., shake carefully. Let stand for 15 min at Temp. Ambient. 4. During the incubation period change the medium in the test boxes: aspirate the old medium and replace with 1.5 ml of medium (containing 5% FCS).

. Place the plasmid-TR mixture of 3 (approximately 320 μl) in the fresh medium and distribute equally by carefully stirring the box.

Incubate for 6 h in an incubator (at 37%, C02 at 5%).

Then supplement the medium to a final FCS concentration of 5-10%. Final volume of the medium of 6 mi.

Incubate for 18 h in an incubator.

Then fully aspirate the mixture and replace with 5 ml of the medium containing FCS at 5 - 10%. cellular sis Aspirate the medium and wash the cells three times with PBS cooled with ice.

Carefully aspirate residual fluid and add 1 ml of lysis buffer per box. Incubate for 30 min at Temp. Atmosphere, let the boxes rest without agitation.

Remove the cell lysate by pipette and transfer to 1.5 ml Eppendorf tubes. 4. Centrifuge the lysates in a bank centrifuge, 3 min.

. Remove the supernatant and transfer to a fresh container. Discard the sediment. 4. Determination of protein from lysates The determination of protein is carried out after lysis. The determination of the protein is analyzed according to Bradford (M. Bradford, Anal. Biochem. 72, 248 (1976)). For further examination, the lysates of an experiment are adjusted with sample buffer (from CAT-ELISA, Boehringer mannhein, order No. 1363727) to the same protein concentration (approximately 250 μg / ml).

. Determination of transfection efficiency The lysates are examined in a CAT-ELISA (Boehringer Mannheim, Order No. 1363727). 200 μl of each of the lysates adjusted to the same protein concentration are used in the ELISA.

The comparison of the absorbances produces the information about the transfection efficiencies.

Reconstitution of equipment components and dilution to work concentration: 1. Normal CAT, bottle 1 Add 0.5 ml of redistilled water to a freeze-dried bottle and dissolve by agitation. The concentration (ng / ml) is printed on a label.

Work dilution: Dilute the dissolved lyophilisate (0.2 mg / ml) with diluent buffer of the sample (bottle 7) to 2 μg / ml. 3. PAB < Dig > POD, bottle 3 Add 0.5 ml of redistilled water to a bottle of lyophilisate and dissolve by agitation (concentration = 20 U / ml).

Work dilution: Dilute the dissolved lyophilisate (20 U / ml) with diluent buffer of the sample (bottle 7) to 150 μg / ml. 4. Shock absorber, bottle 6 Prepare the wash buffer ready for use by mixing 1 part 10 x wash buffer (bottle 6) with 9 parts redistilled water. 1 x wash buffer is required for all washing steps.

. Shock absorber sample, bottle 7 Carrier bottle No. 7 a temp. Environment in a water bath. Ready to use 6. Substrate of POP, bottle 4 Substrate of 1 component of ABTS (bottle No. 4), ready to use, temperature Maintains constant at temp. Ambient.

Process The following table refers to the work dampers and dilutions mentioned above. The MTP modules are clogged in multiple layers of cellulose paper after each washing step.

For incubation steps at 37 ° C, an MTP cover sheet is adhered to the plate.

The results are shown in Figures 1 and 2.

It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property:

Claims (14)

1. Compounds of the general formula (I) characterized in that Ri represents a chain of saturated or unsaturated C (0) -C? -23 or saturated or unsaturated C? -24, Ai represents a group 0-R2 in which R2 has the meaning indicated for Ri and can be the same as, or different from Ri. A2 represents an NR3X or a residue of N + R3R4R5Y "in which R3, R4 can be the same as, or different from, each other, provided that R3 and R can not simultaneously be hydrogen, they represent hydrogen, a group alkyl with 1 to 4 carbon atoms, a group (CH2) n-0H or (CH2) n-NH2, where n = 2-6, R5 which may be the same or different as R3 or R4, denotes hydrogen, a group alkyl having 1 to 4 carbon atoms, a (CH2) n-OH, a (CH2) n-halogenide, or a group ((CH2) mNH) s- (CH2) n-NH2, in which m is a number whole from 2 to 6 and can be equal to, or different year where n can be a number from 2 to 6 and an integer from 0 to 4, X can, in addition to the meaning for R5, have the following meaning: an amino acid amidically linked, an amidically linked peptide or polypeptide, a group C (0) -CHR6N (R) 2, C (0) CHR6N + (R7) 3, C (0) -CHR6N + (Rt) 2R8 or C (0) -CHR6NR7R8 with the proviso that X denotes an amidically bound amino acid, a derivative of suitable amino acids, a peptide or polypeptide if R3 is hydrogen, wherein R6 can be a residue (CH2) m-NR7R8 / (CH2) m- N + (R7) 2R8 or (CH2) m-N + (R7) 3 and m can be be an integer from 1 to 5, R7 represents hydrogen or an alkyl group with 1 to 4 carbon atoms, R8 represents a group (CH2) nN (R7) 2 or (CH2) n- N + (R7) 3 in which n is a number from 2 to 4 and R7 can have the meaning indicated above, and Y is a pharmaceutically acceptable anion, Bi is a residue NH [C (0) - (CH2) P-NH] qZ in which p is a number from 1 to 6 and q is a number from 0 to 2, Z represents an amino acid amidically bound, or an amidically linked peptide or polypeptide, a group C (0) -CHR6N (R7) 2 C (0) -CHR6N + (R7) 3, C (0) - CHR6N + (R7) 2R8 or C (0) -CHR6NRR8, and R6 to R8 and m have the meanings mentioned above, and B2 can have the meaning indicated for i, and the meaning for Ai is only valid with Bx and that of A2 is only valid with B2.
2. 2. The compounds according to claim 1, characterized in that the Rx represents a C 0 0.20 or C (0) 0-2 o saturated or unsaturated group.
3. 3. The compounds according to claim 1 or 2, characterized in that A2 denotes NR3X wherein R3 is hydrogen and X is an amidically linked amino acid, an amino acid derivative, a peptide or polypeptide,. The compounds of the general formula (I) RjO ~ Al, 2 (I) Bl, 2 - characterized because Ri represents a saturated or unsaturated chain of C (0) -C? _23 or saturated or unsaturated C? _24, Ai represents a group 0-R2 in which R2 has the meaning indicated for Ri and can be the same as, or different to Ri. A2 represents an NR3X or a residue of N + R3R4R5Y "in which R3, R4 may be the same as, or different from, each other, represent hydrogen, represent hydrogen, an alkyl group with 1 to 4 carbon atoms, a group (CH2) n-OH or (CH2) n-NH2, where n = 2-6, R5 which may be the same or different as R3 or R4, denotes hydrogen, an alkyl group with 1 to 4 carbon atoms, a (CH2) n-0H, a (CH2) n- halide, or a group ((CH2) mNH) Q- (CH2) n-NH2, in which m is an integer from 2 to 6 and can be equal to , or different year where n can be a number from 2 to 6 and an integer from 0 to 4, X can, in addition to the meaning for R5, have the following meaning: an amidically linked amino acid, an amidically linked peptide or polypeptide, a group C (0) -CHR6N (R7) 2, C (0) CHR6N + (R7) 3, C (0) -CHR6N + (R7) 2R8 or C (0) -CHR6NR7R8 in which R6 can be a residue (CH2) ro-NR7R8, (CH2) m- N + (R -7) 2R8 or (CH2) m-N + (R7) 3 and can be an integer from 1 to 5, R7 represents hydrogen or an alkyl group with 1 to
4. 4 carbon atoms, R8 represents a group (CH2) n-N (R7) 2 or (CH2) n- N + (R7) 3 in which n is a number from 2 to 4 and R7 can have the meaning indicated above, and Y is a pharmaceutically acceptable anion, Bi is a residue NH [C (0) - (CH2) P-NH] qZ in which p is a number from 1 to 6 and q is a number since 0 to 2, Z represents an amidically linked amino acid, or an amidically linked peptide or polypeptide, a C (0) -CHR6N (R7) 2 C (0) -CHR6N + (R7) 3, C (0) -CHR6N + (R7) group ) 2R8 or C (O) -CHR6NR7R8, and R6 to R8 and m have the meanings mentioned above, and B2 can have the meaning indicated for Ax, and the meaning for Ai is only valid with Bi and that of A2 is only valid with B2 .
5. 5. The compounds according to claim 1, 2, 3 or 4, characterized in that Y denotes a halide, monomethyl sulfate, acetate, trifluoroacetate, or phosphate.
6. 6. The compounds according to one of claims 1 to 5, characterized in that it is 2- (6-carboxypermyl) -1,3-dioleoyloxy-propylamide, 1- (6-carboxypermyl) -2,3-dioleoyloxy-propylamide, 2, 3-Daryoyloxy-N- (N- (6-carboxypermyl) -glycyl) -aminopropane, 2- (6-carboxypermyl) -1,3-dyristoxy-propylamide, 2, (1,1,1,5,5) , 10,10,14,14, 14-deca-methyl-6-carboxyespermil) -1, 3-dioleoxyloxy-propylamide 2- (N, N, N, N ', N', N '-hexamethylornityl) -1, 3-Dioleoyloxy-propylamide and / or 2- (,, N, N, N ', N', N '-hexamethyllysyl) -1,3-dioleoyloxy-propylamide.
7. 7. Reagent composed of at least one compound of the general formula (I), characterized in that the solution contains a water-miscible solvent and the formula (I) is defined as follows: Bl, 2 - where: Ri represents a saturated or unsaturated C (0) -C? -23 chain or saturated or unsaturated C? -24, Ai represents a group 0-R2 in which R2 has the meaning indicated for Ri and can be the same as, or different from Ri. A2 represents an NR3X or a residue of N + R3RR5Y "in which R3, R4 may be the same as, or different from, each other, represent hydrogen, represent hydrogen, an alkyl group with 1 to 4 carbon atoms, a group (CH2) n-OH or (CH2) n-NH2, where n = 2-6, R5 which may be the same or different as R3 or R4, denotes hydrogen, an alkyl group with 1 to 4 carbon atoms, a (CH2) n-OH, a (CH2) n-halide, or a group ((CH2) mNH) 0- (CH2) n-NH2, in which m is an integer from 2 to 6 and can be equal to , or different year where n can be a number from 2 to 6 and an integer from 0 to 4, X can, in addition to the meaning for R5, have the following meaning: an amidically linked amino acid, an amidically linked peptide or polypeptide, a group C (O) -CHR6N (R7) 2, C (0) CHR6N + (R7) 3, C (0) -CHR6N + (R7) 2R8 or C (O) -CHR6NR7R9 in which R6 can be a residue (CH2 ) m-NR7R8, (CH2) m- N + (R7) 2R8 or (CH2) m-N + (R7) 3 and m can be an integer from 1 to 5, R represents hydrogen or an alkyl group with 1 to 4 carbon atoms, R8 represents a group (CH2) nN (R7) 2 or (CH2) n- N + (R7) 3 in which n is a number from 2 a 4 and R7 can have the meaning indicated above, and Y is a pharmaceutically acceptable anion, Bi is an NH [C (O) - (CH2) P-NH] qZ residue in which p is a number from 1 to 6 and q is a number from 0 to 2, Z represents an amino acid amidically bound, or an amidically linked peptide or polypeptide, a group C (0) -CHR6N (R7) 2 C (0) -CHR6N + (R7) 3, C (0) - CHR6N + (R7) 2Ra or C (0) -CHR6NR7R8, and R6 to R8 and m have the meanings mentioned above, and B2 can have the meaning indicated for Ai, and the meaning for Ai is only valid with Bx and that of A2 is only valid with B2.
8. 8. The reagent according to claim 7, characterized in that the solution contains a mixture composed of at least one compound of the general formula (I) and another lipid compound in water miscible solvents.
9. 9. The reagent according to claim 7 or 8, characterized in that the solvent miscible in water is a lower alcohol.
10. 10. The reagent characterized in that one or more compounds as claimed in claim 1 to 6 are present in the form of liposomes or other aggregates optionally in the presence of lipid compounds.
11. 11. A method for the distribution of (bio) molecules in cells, characterized in that a reagent of claims 7 to 10 is used and this is first contacted with an anionic biomolecule and subsequently carried out with cells for transfection.
12. 12. The method according to claim 11, characterized in that the molecules are transported with DNA or RNA.
13. 13. The method according to claim 11, characterized in that the anionic biomolecule is DNA or a corresponding fragment.
14. 14. The method according to claim 11 to 13, characterized in that it is a 2- (6-carboxypermyl) -1,3-dioleoyloxy-propylamide, 1- (6-carboxypermyl) -2,3-dioleoyloxy-propylamide, 2, 3 -Daryloyloxy-N- (N- (6-carboxypermyl) -glycyl) -aminopropane, 2- (6-carboxypermyl) -1,3-dyristoxy-propylamide, 2, (1,1,1,5,5,10, 10,14,14, 14-deca-methyl-6-carboxyespermil) -1, 3-dioleoxyloxy-propylamide 2- (N, N, N, N ', N', N '-hexamethylornityl) -1,3-dioleoyloxy -propylamide and / or 2- (, N, N,, N ', N', N '-hexamethillisyl) -1, 3-dioleoyloxy-propylamide.