Branched amphiphilic lipids

Abstract

Disclosed are novel branched amphiphilic lipids, in particular novel branched amphiphilic lipids of general formula (II). Also disclosed is a method of synthesis for the compounds of formula (II), from unsaturated amphiphilic compounds of general formula (I). Further disclosed is the use of the compounds of general formula (II) and of the lipoplexes obtained by formulation of the compounds of general formula (II) for applications, particularly transfection, in which improved fusion properties are desired. ##STR00001##

Claims

1. An amphiphilic compound of general formula (II): ##STR00044## wherein: L.sub.1 and L.sub.2 are each independently a linker selected from the group consisting of a single bond and alkyl; L.sub.3 is a linker selected from the group consisting of alkylphosphoramidates and alkyloxy; Z is a polar functional group, said group being cationic, anionic, zwitterionic or neutral; a is 1; n, n, q and q are each independently an integer from 1 to 15; m, m, p and p are each independently an integer from 0 to 4 with the condition that: at least one of m and p is different from 0; and at least one of m and p is different from 0; R.sub.1 and R.sub.2 are one a hydrogen and the other a thioether group of formula S-L.sub.4-R.sub.5 and R.sub.3 and R.sub.4 are one a hydrogen and the other a thioether group of formula S-L.sub.4-R.sub.5 wherein: L.sub.4 is a linker selected from the group consisting of a single bond, alkyl, cycloalkyl and alkylaryl; and R.sub.5 is a hydrogen atom, or: polar group selected from the group consisting of organic salts of the ammonium, phosphonium, and imidazolium type and protonable neutral heterocycles; a reactive group selected from the group consisting of group N.sub.3, amino, alkylamino, COOH, amide, maleimide, alkyne, SH, OH, ester, activated ester, activated carboxylic acid, halo, nitro, nitrile, isonitrile, acrylamide, aldehyde, ketone, acetals, ketals, anhydride, thiocyanate, isothiocyanate, isocyanate, hydrazine, hydrazides, hydrazones, ethers, oxides, cyanates, diazo, diazonium, sulphides, disulphides, sulphoxides, sulphones, sulphonic acids, sulphinic acids, sulphates, sulphenic acids, amidines, imides, imines, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids, thiohydroxamic acids, allenes, ortho esters, sulphites, enamines, ynamines, ureas, pseudo-ureas, semicarbazides, carbodiimides, and carbamates; or a bioactive group selected from the group consisting of amino acids, peptides, proteins, antibodies, enzymes, polysaccharides, nucleosides, nucleotides, oligonucleotides, fluorophores, chromophores, radioisotopes, carboranes.

2. The compound according to claim 1, of formula (IIa): ##STR00045## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, Z, n, n, m, m, p, p, q and q are as defined in claim 1; R.sub.6 is a hydrogen or an alkyl; and r is an integer from 1 to 10.

3. The compound according to claim 1, of formula (IIb): ##STR00046## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, Z, n, n, m, m, p, p, q and q are as defined in claim 1; and s is an integer from 1 to 10.

4. The compound according to claim 1, selected from the group consisting of: ##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051## wherein X.sup. is a counterion.

5. A liposome comprising at least one of the compounds according to claim 1 having a hexagonal phase.

6. A lipoplex comprising at least one of the compounds according to claim 1 having a hexagonal phase.

7. A pharmaceutical composition comprising a compound according to claim 1 and a physiologically acceptable vehicle.

8. A pharmaceutical composition comprising a liposome according to claim 5 and a physiologically acceptable vehicle.

9. A pharmaceutical composition comprising a lipoplex according to claim 6 and a physiologically acceptable vehicle.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a set of NMR .sup.31P spectra (Hahn echo) of the compounds I-1, II-1, II-2, II-3, II-4 and II-8 (in a mixture with the compound I-1 with a ratio 1/1) carried out at ambient temperature.

(2) FIG. 2 relates to the evaluation of the condensation of DNA by the various compounds of the invention, and formulated either alone () or with DOPE (+). A is an agarose gel 1% after migration (0.1 g of DNA per well; B is a graph showing the fluorescence of the low band of DNA in relation to the charge ratio. The fluorescence of the low band of DNA was quantified then expressed as a percentage of the maximum fluorescence, measured for non-complexed naked DNA.

(3) FIG. 3 is a bar graph showing the transfection effectiveness in vitro of cells C2C12 by means of compounds of the invention and controls the compound I-1 and DOTMA. The threshold for positivity (dashed line) is determined with non-transfected cells (exposed to non-complexed naked DNA; DNA). CR, charge ratio.

(4) FIG. 4 is a bar graph showing the viability of C2C12 cells after transfection in vitro by means of compounds of the invention and controls the compound I-1 and DOTMA. The viability of non-transfected cells (exposed to non-complexed naked DNA; DNA) is used as a reference (value 100%, dashed lines). CR, charge ratio.

(5) FIG. 5 is a bar graph showing the transfection effectiveness and cell viability of compounds with branched fatty chains expressed in relation to cells obtained with the compound with linear fatty chains. For each compound, the values considered were those obtained at the charge ratio for which the peak in effectiveness was reached.

EXAMPLES

(6) This invention will be understood better when reading the following examples which show the invention in a non-limiting way.

SYNTHESIS

Abbreviations

(7) DNA: deoxyribonucleic acid; RNA: ribonucleic acid; siRNA: small interfering RNA; mRNA: messenger RNA; C.: degrees Celsius; DOTMA: N-[1-(2,3-dioleyloxy)propylJ-N,N,N-trimethylammonium chloride; DCC: N,N-dicyclohexylcarbodiimide; DMEM: Dulbecco's modified Eagle's essential minimum medium; eq.: equivalent; EGFP-Luc: Enhanced green fluorescent protein-luciferase; s: microsecond; Ppm: parts per million; CR: charge ratio; NMR: nuclear magnetic resonance; TMS: tetramethylsilane; UV: ultra-violet.

(8) Materials

(9) The solvents used were purified according to the usual methods.

(10) The starting substrates were supplied by Aldrich, TCI or Alfa Aesar and used without any additional purification. The 2-((bis(E)-octadec-9-en-1-yloxy)phosphoryl)amino)-N,N,N-trimethylthanaminium iodide (reagent I-1) was synthesised according to the literature. (Le Corre, S. S.; Berchel, M.; Belmadi, N.; Denis, C.; Haelters, J. P.; Le Gall, T.; Lehn, P.; Montier, T.; Jaffrs, P. A. Org. Biomol. Chem., 2014, 12, 1463-1474.).

(11) ##STR00040##

(12) All of the compounds were characterised by nuclear magnetic resonance spectroscopy (NMR).sup.1H (500.13 or 400.133 or 300.135 MHz), .sup.13C (125.773 or 75.480 MHz) and .sup.31P (161.970 or 121.498 MHz) (Bruker AC 300, Avance DRX 400 and Avance DRX 500 Spectrometers). The J coupling constants are given in Hertz. The following abbreviations are used: s for singlet, d for doublet, t for triplet, q for quadruplet, qt for quintuplet, m for multiplet, dd for doublet of doublets and dt for doublet of triplets.

(13) The compounds were also characterised by mass spectrometry (Bruker Autoflex MALDI TOF-TOF III LRF200 CID).

(14) Results

(15) 1) Synthesis of Zwitterionic Reagents

(16) ##STR00041##

(17) To a solution of dioleyl phosphoramide (1 eq) in the chloroform, is added propanesulfone (1.2 eq). The reaction medium is stirred for 72 h at ambient temperature. The raw product obtained is then purified by flash chromatography on a silica gel column.

(18) The following compound was synthesised according to the method described hereinabove.

(19) Compound I-2: Yield: 44%, .sup.31P NMR: (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm in CDCl.sub.3): 9.4; .sup.1H NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 0.85 to 0.89 (t, .sup.3J.sub.HH=6.8 Hz, 6H, CH.sub.3CH.sub.3), 1.21 to 1.29 (m, 46H, CH.sub.2 fatty chain), 1.62 to 1.65 (m, 4H, CH.sub.2CH.sub.2OP), 1.96 to 2.01 (m, 8H, CH.sub.2CHCHCH.sub.2), 2.19 to 2.26 (m, 2H, .sup.+NCH.sub.2CH.sub.2CH.sub.2S), 2.89 to 2.91 (m, 2H, .sup.+NCH.sub.2CH.sub.2CH.sub.2S), 3.24 (s, 6H, (CH.sub.3).sub.2N.sup.+), 3.41 to 3.47 (m, 2H, .sup.+NCH.sub.2CH.sub.2), 3.51 to 3.61 (m, 2H, NCH.sub.2CH.sub.2-.sup.+NCH.sub.2), 3.67 to 3.72 (m, 2H, NCH.sub.2CH.sub.2.sup.+NCH.sub.2), 3.90 to 3.96 (m, 4H, CH.sub.2OP), 4.82 to 5.00 (m, 1H, PNH), 5.29 to 5.37 (m, 4H, CHCH); .sup.13C NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 14.2 (CH.sub.3CH.sub.2), 25.5 (CH.sub.2 fatty chain), 27.2 (CH.sub.2 fatty chain), 28.9 to 30.5 (CH.sub.2 fatty chain), 31.9 (CH.sub.2 fatty chain), 51.3 ((CH.sub.3).sub.2N.sup.+), 67.0 (CH.sub.2OP), 129.9 to 130.2 (CHCH).

(20) 2) Synthesis of the Compounds of the Invention

(21) 2.1) Click Chemistry with Amphiphilic Lips

(22) ##STR00042##

(23) The compound of formula (I) (1 eq) and the thiol (3.5 eq) are mixed in a glass tube, which is placed in an ultrasound bath until complete dissolution of the compound of formula (I) (10 to 20 minutes). The mixture is degassed under argon, before the adding of 2,2-dimethoxy-2-phenylacetophenone (10% by weight). The solution is placed under UV, at ambient temperature, for 4 hours. The raw product obtained is then purified by flash chromatography on a silica gel column.

(24) The following compounds were synthesised according to the method described hereinabove.

(25) Compounds II-1: Yield: 42% (120 mg); .sup.31P NMR: (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm in CHCl.sub.3)=8.6; .sup.1H NMR: (ppm, reference TMS: 0 ppm in CHCl.sub.3)=0.84 to 0.91 (m, 12H, CH.sub.3CH.sub.2), 1.30 to 1.66 (m, 76H, CH.sub.2 fatty chain), 2.43 to 2.47 (t, .sup.3J.sub.HH=8.0 Hz, 4H, CH.sub.2S), 2.50 to 2.56 (qt, .sup.3J.sub.HH=6 Hz, 2H, (CH.sub.2).sub.2CHS), 3.45 (s, 9H, .sup.+N(CH.sub.3).sub.3), 3.55 to 3.63 (m, 2H, CH.sub.2NH), 3.85 to 3.88 (m, 2H, CH.sub.2.sup.+N(CH.sub.3).sub.3), 3.95 to 3.99 (m, 4H, CH.sub.2OP), 4.40 to 4.53 (m, 1H, NH); .sup.13C NMR: (ppm, reference TMS: 0 ppm in CHCl.sub.3)=13.8 (CH.sub.3CH.sub.2), 22.3 to 31.6 (CH.sub.2 fatty chain), 34.6 (CH.sub.2CHCH.sub.2), 35.9 (CH.sub.2NH), 45.6 (CHS), 54.5 (.sup.+N(CH.sub.3).sub.3), 66.4 (CH.sub.2.sup.+N(CH.sub.3).sub.3), 66.8 (d, .sup.2J.sub.CP=5.2 Hz, CH.sub.2OP); MALDI-TOF: [M].sup.+.sub.calculated for C.sub.53H.sub.112N.sub.2O.sub.3PS.sub.2=919.785, [M].sup.+.sub.measured=919.775;

(26) Compounds II-2: Yield: 65% (150 mg); NMR .sup.31P: 6 (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm in CHCl.sub.3)=8.6; NMR .sup.1H: 6 (ppm, reference TMS: 0 ppm in CHCl.sub.3)=0.85 to 0.88 (t, .sup.3J.sub.HH=6.4 Hz, 12H, CH.sub.3CH.sub.2), 1.24 to 1.66 (m, 76H, fatty chain), 2.43 to 2.47 (t, .sup.3J.sub.HH=8.0 Hz, 4H, CH.sub.2S), 2.51 to 2.55 (qt, .sup.3J.sub.HH=8.0 Hz, 2H, (CH.sub.2).sub.2CHS), 3.45 (s, 9H, .sup.+N(CH.sub.3).sub.3), 3.50 to 3.61 (m, 2H, CH.sub.2NH), 3.83 to 3.86 (m, 2H, CH.sub.2.sup.+N(CH.sub.3).sub.3), 3.95 to 4.00 (m, 4H, CH.sub.2OP), 4.30 to 4.43 (m, 1H, NH); NMR .sup.13C: (ppm, reference TMS: 0 ppm in CHCl.sub.3)=13.9 (CH.sub.3CH.sub.2), 22.5 ppm to 31.8 (CH.sub.2 fatty chain), 34.8 (CH.sub.2CHCH.sub.2), 36.0 (CH.sub.2NH), 45.8 (CHS), 54.7 (.sup.+N(CH.sub.3).sub.3), 66.6 (CH.sub.2.sup.+N(CH.sub.3).sub.3), 66.9 (d, .sup.2J.sub.CP=5.3 Hz, CH.sub.2OP); MALDI-TOF: [M].sup.+.sub.calculated=1087.975; [M].sup.+.sub.measured for C.sub.65H.sub.136N.sub.2O.sub.3PS.sub.2=1087.972;

(27) Compound II-3: Yield: 58% (150 mg); .sup.31P NMR: (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm in CHCl.sub.3)=8.7; .sup.1H NMR: (ppm, reference TMS: 0 ppm in CHCl.sub.3)=0.83 to 0.91 (m, 6H, CH.sub.3CH.sub.2), 1.25 to 1.68 (m, 60H, fatty chain), 2.45 to 2.51 (qt, J.sub.HH=6.4 Hz, 2H, (CH.sub.2).sub.2CHS), 3.43 (s, 9H, .sup.+N(CH.sub.3).sub.3), 3.48 to 3.52 (m, 2H, CH.sub.2NH), 3.68 (s, 4H, CH.sub.2-Ph), 3.83 to 3.86 (m, 2H, CH.sub.2.sup.+N(CH.sub.3).sub.3), 3.96 to 4.01 (m, 4H, CH.sub.2OP), 4.30 to 4.45 (m, 1H, NH), 7.19 to 7.33 (m, 10H, CH aromatic); .sup.13C NMR: (ppm, reference TMS: 0 ppm in CHCl.sub.3)=14.1 (CH.sub.3CH.sub.2), 22.6 to 31.8 (CH.sub.2 fatty chain), 34.5 ((CH.sub.2).sub.2CHS), 35.0 (CH.sub.2-Ph), 45.3 ((CH.sub.2).sub.2CHS), 54.8 ((CH.sub.3).sub.3N.sup.+), 66.6 (CH.sub.2.sup.+N(CH.sub.3).sub.3), 67.1 (d, .sup.2J.sub.PC=5.3 ppm, CH.sub.2OP), 126.7 (CH aromatic), 128.3 to 129.0 (CH aromatic), 138.9 (quaternary C); MALDI-TOF: [M].sup.+.sub.calculated for C.sub.55H.sub.100N.sub.2O.sub.3PS.sub.2=931.691; [M].sup.+.sub.measured=931.686;

(28) Compound II-4: Yield: 51% (133 mg); .sup.31P NMR: (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm in CHCl.sub.3)=8.6; .sup.1H NMR: (ppm, reference TMS: 0 ppm in CHCl.sub.3)=0.81 to 0.87 (t, .sup.3J.sub.HH=8 Hz, 6H, CH.sub.3CH.sub.2), 1.24 to 1.90 (m, 80H, fatty chain), 2.58 to 2.62 (m, 4H, CHSCH), 3.53 (s, 9H, .sup.+N(CH.sub.3).sub.3), 3.50 to 3.60 (m, 2H, CH.sub.2NH), 3.83 to 3.85 (m, 2H, CH.sub.2.sup.+N(CH.sub.3).sub.3), 3.93 to 3.98 (m, 4H, CH.sub.2OP), 4.30 to 4.40 (m, 1H, NH); .sup.13C NMR: (ppm, reference TMS: 0 ppm in CHCl.sub.3)=14.1 (CH.sub.3CH.sub.2), 22.6 to 31.9 (CH.sub.2 fatty chain), 34.3 (CH.sub.2 cyclohexyl), 35.5 ((CH.sub.2).sub.2CHS), 36.2 (CH.sub.2NH), 42.6 (CH cyclohexyl), 44.2 ((CH.sub.2).sub.2CHS), 54.9 (.sup.+N(CH.sub.3).sub.3), 66.7 (CH.sub.2.sup.+N(CH.sub.3).sub.3), 67.1 (POCH.sub.2); MALDI-TOF: [M].sup.+.sub.calculated for C.sub.53H.sub.108N.sub.2O.sub.3PS.sub.2=915.753; [M].sup.+.sub.measured=915.741;

(29) Compound II-5: Yield: 61% (199 mg); .sup.1H NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3)=0.83 to 0.87 (t, .sup.3J.sub.HH=6.6 Hz, 12H), 1.23 to 1.24 (m, 73H, CH.sub.2 fatty chain), 1.35 to 1.37 (m, 12H, CH.sub.2 fatty chain), 1.46 to 1.54 (m, 16H, CH.sub.2CH.sub.2S, CH.sub.2CHS and CH.sub.2 fatty chain), 2.41 to 2.45 (t, .sup.3J.sub.HH=7.4 Hz, 4H, CH.sub.2S), 2.48 to 2.55 (qt, .sup.3J.sub.HH=7.3 Hz, 2H, (CH.sub.2)CHS), 3.37 to 3.56 (m, 15H, OCH.sub.2 and .sup.+N(CH.sub.3).sub.3), 3.65 to 3.69 (m, 1H, OCH), 3.95 to 3.99 (m, 2H, OCH.sub.2); .sup.13C NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3)=14.1 (CH.sub.3CH.sub.2), 22.7 (CH.sub.2 fatty chain), 26.0 (CH.sub.2 fatty chain), 26.2 (CH.sub.2 fatty chain), 26.8 (CH.sub.2 fatty chain), 29.0 to 29.9 (CH.sub.2 fatty chain), 30.35 (CH.sub.2SCH), 31.90 (CH.sub.2 fatty chain), 34.91 (CH.sub.2 fatty chain), 46.88 ((CH.sub.2).sub.2CHS), 54.79 (.sup.+N(CH.sub.3).sub.3), 67.93 (OCH.sub.2), 68.38 (OCH.sub.2), 69.31 (OCH.sub.2), 72.01 (OCH.sub.2), 73.63 (OCH);

(30) Compound II-6: Yield: 48% (140 mg); .sup.31P NMR: (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm in CDCl.sub.3): 8.3; .sup.1H NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 0.85 to 0.89 (t, .sup.3J.sub.HH=8 Hz, 6H, CH.sub.3CH.sub.2), 1.24 to 1.56 (m, 75H, CH.sub.2 fatty chain), 1.49 to 1.56 (m, 12H, CH.sub.2 fatty chain), 1.63 to 1.66 (m, 4H, CH.sub.2CH.sub.2O), 1.95 to 2.06 (m, 4H, CH.sub.2CH.sub.2OH), 2.43 to 2.47 (t, .sup.3J.sub.HH=7.2 Hz, 4H, CH.sub.2S), 2.51 to 2.54 (m, 2H, CHS), 3.44 (s, 9H, (CH.sub.3).sub.3N.sup.+), 3.48 to 3.58 (m, 2H, .sup.+NCH.sub.2CH.sub.2), 3.59 to 3.63 (t, .sup.3J.sub.HH=6.8 Hz, 4H, CH.sub.2OH), 3.85 to 3.88 (m, 2H, CH.sub.2OP), 3.96 to 4.01 (m, 4H, CH.sub.2OP), 4.51 to 4.62 (m, 1H, NHP); 13C NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 14.1 (CH.sub.3CH.sub.2), 22.6 to 36.0 (CH.sub.2 fatty chain), CH.sub.2S), 39.2 (CH.sub.2NHP), 45.9 ((CH.sub.2).sub.2CHS), 54.8 (.sup.+N(CH.sub.3).sub.3), 62.8 (CH.sub.2CH.sub.2OH), 66.5 (CH.sub.2.sup.+N(CH.sub.3).sub.3), 67.2 to 67.3 (d, .sup.2J.sub.HH=22 Hz, CH.sub.2OP);

(31) Compound II-7: Yield: 23% (65 mg); .sup.31P NMR: (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm in CDCl.sub.3): 8.2; .sup.1H NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 0.86 to 0.89 (t, .sup.3J.sub.HH=6.4 Hz, 6H, CH.sub.3CH.sub.2), 1.24 to 1.37 (m, 68H, CH.sub.2 fatty chain), 1.46 to 1.66 (m, 16H, CH.sub.2 fatty chain), 2.29 to 2.40 (m, 4H, CH.sub.2CH.sub.2OH), 2.44 to 2.47 (t, .sup.3J.sub.HH=7.2 Hz, 4H, CH.sub.2S), 2.51 to 2.53 (m, 2H, CHS), 3.45 (s, 9H, (CH.sub.3).sub.3N.sup.+), 3.47 to 3.54 (m, 2H, .sup.+NCH.sub.2CH.sub.2), 3.59 to 3.63 (t, .sup.3J.sub.HH=6.8 Hz, 4H, CH.sub.2OH), 3.86 to 3.88 (m, 2H, CH.sub.2NHP), 3.97 to 4.02 (m, 4H, CH.sub.2OP), 4.51 to 4.62 (m, 1H, NHP); .sup.13C NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 14.1 (CH.sub.3CH.sub.2), 22.7 to 36.1 (CH.sub.2 fatty chain and CH.sub.2S and CH.sub.2CH.sub.2OH), 45.9 (CHS), 54.9 ((CH.sub.3).sub.3N.sup.+), 62.8 (CH.sub.2OH), 66.5 (CH.sub.2NHP), 67.4 to 67.5 (d, .sup.2J.sub.PC=21 Hz, CH.sub.2OP);

(32) Compound II-8: Yield: 51%, .sup.31P NMR: (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm in CDCl.sub.3): 9.5, .sup.1H NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 0.86 to 0.88 (t, .sup.3J.sub.HH=6.8 Hz, 12H, CH.sub.3CH.sub.2), 1.24 to 1.57 (m, 89H, CH.sub.2 fatty chain), 1.63 to 1.64 (m, 4H, CH.sub.2CH.sub.2OP), 2.18 to 2.26 (m, 2H, .sup.+NCH.sub.2CH.sub.2CH.sub.2S), 2.42 to 2.46 (t, .sup.3J.sub.HH=7.4 Hz, 4H, CH.sub.2SCH(CH.sub.2).sub.2), 2.49 to 2.54 (st, .sup.3J.sub.HH=6.2 Hz, 2H, CH.sub.2SCH(CH.sub.2).sub.2), 2.88 to 2.90 (m, 2H, CH.sub.2SO), 3.22 (s, 6H, (CH.sub.3).sub.2N.sup.+), 3.42 to 3.47 (m, 2H, CH.sub.2NH), 3.50 to 3.60 (m, 2H, PNHCH.sub.2), 3.66 to 3.69 (m, 2H, .sup.+NCH.sub.2CH.sub.2CH.sub.2S), 3.91 to 3.95 (m, 4H, CH.sub.2CH.sub.2OP), 4.81 to 4.91 (m, 1H, PNH); .sup.13C NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 14.1 (CH.sub.3CH.sub.2), 22.7 (CH.sub.2 fatty chain), 25.7 (CH.sub.2 fatty chain), 26.8 (CH.sub.2 fatty chain), 27.0 (CH.sub.2 fatty chain), 29.1 to 30.0 (CH.sub.2 fatty chain), 30.4 (CH.sub.2SCH(CH.sub.2).sub.2), 31.9 (CH.sub.2 fatty chain), 34.9 to 35.1 (CH.sub.2 fatty chain), 46.0 (CH.sub.2SCH(CH.sub.2).sub.2), 51.5 ((CH.sub.3).sub.2N.sup.+), 66.9 (CH.sub.2OP);

(33) Compound II-9: .sup.31P NMR: (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm in CDCl.sub.3): 10.1, .sup.1H NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 0.85 to 0.88 (t, .sup.3J.sub.HH=6 Hz, 6H, CH.sub.3CH.sub.2), 1.27 to 1.55 (m, 56H, CH.sub.2 fatty chain), 1.62 to 1.37 (m, 4H, CH.sub.2CH.sub.2OP), 1.79 to 1.85 (m, 4H, CH.sub.2SCH(CH.sub.2).sub.2), 1.99 to 2.14 (m, 2H, HOCH.sub.2CH.sub.2CH.sub.2S), 2.21 (s, 6H, (CH.sub.2).sub.2N), 2.36 to 2.39 (t, .sup.3J.sub.HH=5.8 Hz, 2H, CH.sub.2SCH(CH.sub.2).sub.2), 2.55 to 2.61 (m, 6H, HOCH.sub.2CH.sub.2CH.sub.2S and CH.sub.2CH.sub.2N), 2.92 to 2.98 (m, 2H, PNHCH.sub.2), 3.20 to 3.31 (m, 1H, PNHCH.sub.2), 3.71 to 3.76 (m, 4H, HOCH.sub.2CH.sub.2CH.sub.2S), 3.93 to 3.99 (m, 4H, CH.sub.2OP).

(34) 2.2) Functionalisation by a Bioactive Group

(35) ##STR00043##

(36) The ester RC(O)OH (2 eq) and the branched compound II (1 eq) are mixed in the anhydrous dichloromethane. After adding DCC (2.2 eq), the reaction medium is stirred for one night. The compound obtained is purified by flash chromatography on a silica gel column.

(37) The following compound was synthesised according to the method C.

(38) Compound II-10: Yield: 15%, .sup.31P NMR: (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm in CDCl.sub.3): 10.1; .sup.1H NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 0.85 to 0.89 (m, 18H, CH.sub.3CH.sub.2 and (CH.sub.3).sub.2CH), 1.26 to 1.48 (m, 68H, CH.sub.2 fatty chain and CH.sub.3CH-Ph), 1.63 to 1.66 (m, 4H, CH.sub.2CH.sub.2O), 1.79 to 1.84 (m, 4H, SCH.sub.2CH.sub.2CH.sub.2O), 2.24 (s, 6H, (CH.sub.3).sub.2N), 2.38 to 2.45 (m, 12H, CH.sub.2C(O)OCH.sub.2, CH.sub.2S and CHS and CH.sub.2N(CH.sub.3).sub.2), 3.89 to 3.02 (m, 4H, CH.sub.2NHP), 3.34 to 3.42 (m, 1H, NHP), 3.66 to 3.68 (m, 2H, CH.sub.3CH-Ph), 3.94 to 3.96 (m, 4H, CH.sub.2OP), 4.12 to 4.15 (t, .sup.3J.sub.HH=6.3 Hz, 4H, CH.sub.2OC(O)CH.sub.2), 7.06 to 7.08 (d, .sup.3J.sub.HH=8 Hz, 4H, H.sup.2H.sup.6/H.sup.2 H.sup.6), 7.17 to 7.19 (d, .sup.3J.sub.HH=8 Hz, 4H, H.sup.3H.sup.5/H.sup.3 H.sup.5); .sup.13C NMR: (ppm, reference TMS: 0 ppm in CDCl.sub.3): 13.4 (CH.sub.3CH.sub.2), 17.5 (CH.sub.3CH-Ph), 22.4 ((CH.sub.3).sub.2CH), 22.65 (CH.sub.2 fatty chain), 25.6 (CH.sub.2 fatty chain), 26.5 to 26.7 (CH.sub.2 fatty chain and CH.sub.2S), 29.0 to 31.9 (CH.sub.2 fatty chain), 34.8 (CH.sub.2 fatty chain), 38.4 (CH.sub.2NHP), 44.8 to 46.0 (CH.sub.2N(CH.sub.3).sub.2 and CH.sub.2N(CH.sub.3).sub.2 and CHCH.sub.3), 59.5 (CHS), 63.3 (CH.sub.2OC(O)CH.sub.2), 66.3 (CH.sub.2OP), 127.1 (C.sup.3C.sup.5/C.sup.3 C.sup.5), 129.3 (C.sup.2C.sup.6/C.sup.2 C.sup.6), 137.7 (C.sup.4/C.sup.4), 140.4 (C.sup.1/C.sup.1), 174.6 (CH.sub.2C(O)OCH.sub.2).

(39) Studies of the Physical-Chemical Properties of Liposomes and Lipoplexes

(40) 1) Structural OrganisationStudy Via NMR .sup.31P

(41) Preparation of the Sample

(42) A stock solution of a cationic lipid is prepared in the chloroform. A volume of stock solution corresponding to a weight in lipids between 50 mg and 100 mg is pipette into a haemolysis tube and the chloroform is evaporated under of flow of N.sub.2 in such a way as to obtain a lipid film. 500 L of water is then added and the mixture is subjected to ultrasound until complete dispersion of the lipid film. The mixture is lysed for one night. A volume of distilled water is then added in order to obtain a solution with a concentration of 100 mg.Math.mL.sup.1. In order to ensure the balance of the sample, three cycles 198 C./50 C. are carried out: the sample is placed at 198 C. (liquid nitrogen) for 5 min, then the sample is placed in a water bath at 50 C. for 30 min and is vortexed.

(43) Condition for Acquisition

(44) The NMR .sup.31P spectra were acquired with a Hahn echo sequence (90--180--acq). The acquisition parameters were as follows: spectral window of 200 KHz, a pulse /2 with a duration of 4.88 is, a recycling period of 5 s and an echo period of 40 s. The number of acquisitions depends on the volume of the sample and the acquisition temperature, and is between 128 and 1700 scans. The temperature of the sample is balanced for 30 min before the start of the acquisition. Lorentzian noise is filtered over the entire spectral window before the Fourier transform on the top of the echo. Dipalmitoylphosphatidylcholine vesicles (DPPC) act as external reference in the NMR .sup.31P. The deconvolution of the spectra was carried out with the TOPSPIN software and dmfit-2009 (Massiot D, Fayon F, Capron M, King I, Le Calv S, Alonso B, et al. Magn. Reson. Chem., 2002, 40, 70-76).

(45) Results

(46) FIG. 1 very clearly shows via NMR .sup.31P at ambient temperature (Hahn echo sequence) that the unbranched compound (I-1) is self-organised into a hydrated medium in lamellar form while the unbranched compounds of the invention (branching obtained by a reaction of the thiol-ene type; Compounds II-1, II-2, II-3, II-4) generate supramolecular aggregates adopting a hexagonal structure. The NMR .sup.31P spectra, recorded under the same conditions, the co-formulation 1/1 (molar ratio) of the unbranched cationic compound I-1 and of the zwitterionic and branched co-lipid II-8, shows that the co-lipid leads to the formation of supramolecular aggregates of hexagonal structure. This structuring can therefore be induced by the presence in the co-formulation of a neutral lipid (zwitterionic) branched by thiol-ene reaction.

(47) 2) Measurement of the Size and of the Zeta Potential

(48) Preparation of the Samples

(49) The cationic lipids were formulated in a liposomal solution by the method of hydration of a lipid film. Initially, a fraction of a concentrated solution of the cationic lipid is placed in a glass tube and evaporated to give a thin lipid film. 1 mL of water is then added to the film and the film is hydrated for 3 days at 4 C. The solution is then vortexed (1 min) and placed under ultrasound (30 to 60 min) at 45 kHz. Then 150 L of the liposomal solution is diluted in 1.5 mL of sterile water. After filtration (200 m) of the liposomal solution, the size and the zeta potential of liposomes and lipoplexes are determined.

(50) Conditions for Acquisition

(51) The size and zeta potential () of the liposomes and of the lipoplexes were measured with a Zetasizer Nano ZS (Malvern Instruments) at 25 C. after a suitable dilution of the formulations. For the measurements of the lipoplexes, each test used 40 mg of plasmid DNA in water with the required amount of cationic lipid studied in order to form lipoplexes with charge ratios ranging from 0.5 to 8.0. For the measurements of the liposomes, a quantity of lipid equivalent to a mixture having a 1.0 charge ratio in water was used.

(52) Results

(53) TABLE-US-00002 Compound Size (nm) PdI Zeta Potential (mV) PZ (mV) I-1 94 0.26 +54 4 II-1 99 0.26 +33 7 II-2 148 0.15 +37 5 II-3 187 0.36 +44 5 II-4 137 0.19 +50 7 II-8 + I-1 (1/1) 136 0.2 +48 6 II-5 230 0.30 +24 6 II-6 188 0.30 +58 7 II-7 132 0.20 +53 8

(54) This size data shows that the supramolecular aggregates formed according to the method of hydration of a lipid film followed by a sonication step, have very usual sizes between 90 and 230 nm. Polydispersity indices (PdI) reflect the relatively homogeneous nature of these sizes in the samples. The zeta potential of these samples, which is always positive, corresponds to the usual values for cationic amphiphilics or co-formulations comprising at least one cationic amphiphilic.

(55) Biological Study

(56) Materials

(57) Cells and Culture Conditions.

(58) The C2C12 cell line is used as an example; it is immortalised mouse myoblasts. These cells are cultured in DMEM medium (Lonza) supplemented with decomplemented serum (10%), L-Glutamine (2 mM) and penicillin (100 U/mL)+streptomycin (100 g/mL). The cells are maintained in an incubator at 37 C. in a humid atmosphere and 5% CO.sub.2.

(59) Plasmid DNA.

(60) The luciferase plasmid reporter pEGFP-Luc (Clontech) is used in these tests.

(61) Methods Formation of Lipoplexes.

(62) The lipoplexes are prepared in OptiMEM (Gibco) by mixing different amounts of a given compound of the invention with 5 g of DNA, in order to form lipoplexes [lipid/DNA] characterised by charge ratios (+/) between 0.5 and 8.0.

(63) Condensation of DNA.

(64) Ethidium bromide is inserted into the DNA before being mixed with the compounds to be tested. The DNA condensation is then evaluated by agarose gel electrophoresis. DNA alone (not complexed) is used as a reference.

(65) Transfection In Vitro.

(66) The compounds are tested in transient transfection of cultured cells in vitro. The protocol used is one that has been described previously in several scientific publications (Felgner P. L. et al., Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413-7417; Le Gall T. et al., J. Med. Chem. 2010, 53, 1496-1508). Briefly, 24 hours before the actual transfection, the cells are seeded in 96-well plates at a density of 12,500 cells per well. The complexes [lipid/DNA] are deposited directly into the cell culture medium at a rate of 0.25 g of DNA per well. After approximately 36 h of incubation, the culture medium is removed and cells are lysed with 75 L of Passive Lysis Buffer (Promega) 0.5 per well. After 24 h of storage at 20 C., each lysate is used to assay the total protein, measure the luciferase activity and conduct a cell viability test.

(67) Transfection Efficiency.

(68) The luciferase activity is determined with the Luciferase Assay System kit (Promega). Total proteins are quantified using a BCA assay (Interchim) colorimetric test. The transfection efficiency is calculated by dividing the luciferase activity by the quantity of total proteins; it is expressed as relative light units per mg protein (RLU/mg).

(69) Cell Viability.

(70) Cell viability is assessed using the Vialight kit (Lonza). Control cells (untreated) are used as reference (100% viability).

(71) Results

(72) The capacity of compounds to condense DNA is evaluated using the fluorescence properties of the latter when incubated in the presence of ethidium bromide. The condensation of DNA is accompanied by exclusion of the inserted ethidium bromide. The reduction in the fluorescence that results as such provides information on the degree of compaction of the DNA obtained thanks to a given compound.

(73) FIG. 2 shows that the branched compounds of the invention compact the DNA but less complete than the unbranched compounds (I-1).

(74) The lipoplexes are then used in vitro cell transfection. FIG. 3 shows the results obtained by in vitro transfection of C2C12 cells using lipoplexes formed from different branched cationic amphiphilic lipids according to the invention (II-2, II-4 and II-5) or unbranched cationic amphiphilic lipids such as I-1 or DOTMA.

(75) All of the compounds tested are effective for delivering plasmid DNA inside C2C12 cells, cells known to be relatively difficult to transfect. However, compared to the compound with linear fatty chains I-1, compounds with branched fatty chains II-2 and II-4 make it possible to obtain significantly higher transfection efficiencies, with a gain of up to 40 times the reference (FIG. 3). Furthermore, these higher efficiencies are obtained while maintaining good cell viability as shown in FIG. 4.

(76) Compared to DOTMA, the derivative with branched fatty chains II-5 reaches lower transfection efficiencies but it makes it possible to maintain good cell viability, even at high charge ratio (FIG. 5).

(77) These results indicate that a branching of the fatty chains of the cationic lips can make it possible to increase the transfecting power and/or to improve the biocompatibility, i.e. reduce its toxicity, according to the starting cationic lipid and the branching introduced.