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CYCLOHEXANE LIPIDOIDS FOR NUCLEIC ACID TRANSFECTION AND USE THEREOF

20240335559 ยท 2024-10-10

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to a lipidoid of general formula I, wherein X is selected from C(?O)NH, C(?O)O, C(?S)O, C(?O)S, C(?S)S, C(?O)NHNH, CH.sub.2, O, OC(?O), S, SC(?O), NH, NHNH, NHC(?O), NHNHC(?O), C?C, CH?CH, a five-membered heterocycle containing at least 2 nitrogen atoms, CH.sub.2C(?O)NH, CH.sub.2C(?S)O, CH.sub.2C(?S)S, CH.sub.2C(?O)NHNH, N?CH, CH?N, NHN?CH, and CH?NNH; Y is alkylene C.sub.2-C.sub.10 chain; R.sup.1 is selected from alkyl C.sub.1-C.sub.46, alkenyl C.sub.2-C.sub.46, alkynyl C.sub.2-C.sub.46; Z is selected from H, OH, CH.sub.3, CH.sub.2OH, NH.sub.2, C(?O)NH.sub.2, CONH(CH.sub.2).sub.2OH, CON[(CH.sub.2).sub.2OH].sub.2, CONHCH(CH.sub.2OH).sub.2, CONHCH.sub.2CH(OH)CH.sub.2OH, CONH(CH.sub.2).sub.2C(?O)NH.sub.2, CON[CH.sub.2C(?O)NH.sub.2].sub.2, CONH(CH.sub.2).sub.2NHC(?O)NH.sub.2, CONH(CH.sub.2).sub.3N+(CH.sub.3).sub.2(CH.sub.2).sub.2SO.sub.3, CONH(CH.sub.2).sub.3N+(CH.sub.3).sub.2(CH.sub.2).sub.2COO.sup.?, COO(CH.sub.2).sub.2OP(?O)(O)O(CH.sub.2).sub.2N+(CH.sub.3).sub.3, N+(CH.sub.3).sub.2(CH.sub.2).sub.3SO.sub.3, N+(CH.sub.3).sub.2(CH.sub.2).sub.2COO.sup.?, formula (II), and formula (III), wherein R.sup.2 is independently selected from hydrogen and CH.sub.3; E is independently selected from O and S atoms; n is an integer within the range of from 1 to 5; and T is selected from XYN(R.sup.1).sub.2, C(?O)O(C.sub.1-C.sub.3 alkyl), C(?O)OCH.sub.2CH.sub.2OH, formula (IV), formula (V), formula (VI), C(?O)OH, CONH(CH.sub.2).sub.2OH, CON[(CH.sub.2).sub.2OH].sub.2, CONHCH(CH.sub.2OH).sub.2, CONH(CH.sub.2).sub.2C(?O)NH.sub.2, CON[CH.sub.2C(?O)NH.sub.2].sub.2, CONHCH[C(?O)NH.sub.2].sub.2, CONH(CH.sub.2).sub.2NHC(?O)NH.sub.2, C(?O)NH.sub.2, CONH(CH.sub.2).sub.3N+(CH.sub.3).sub.2(CH.sub.2).sub.2SO.sub.3, CONH(CH.sub.2).sub.3N+(CH.sub.3).sub.2(CH.sub.2).sub.2COO.sup.?, NH.sub.2, NHC(?O)CH.sub.3, COO(CH.sub.2).sub.2OP(?O)(O)O(CH.sub.2).sub.2N+(CH.sub.3).sub.3, OH, O(C.sub.1-C.sub.3 alkyl), NHC(?O)NH(CH.sub.3), NHC(?S)N(CH.sub.3).sub.2, NHC(?S)NH(CH.sub.3), NHC(?NCN)NH.sub.2, NHC(?NCN)NH(CH.sub.3), NHC(?NCN)N(CH.sub.3).sub.2, NHC[?NS(?O).sub.2NH.sub.2]NH.sub.2, N+(CH.sub.3).sub.2(CH.sub.2).sub.3SO.sub.3, N+(CH.sub.3).sub.2(CH.sub.2).sub.2COO.sup.?, wherein R.sup.2, E and n are as defined above; and/or if Z is OH or CH.sub.2OH, and T is C(?O)OH, then Z together with T and three carbon atoms between them may form a cyclic lactone containing 4 to 5 carbon atoms; and pharmaceutically acceptable salts, addition salts and solvates thereof. This lipidoid is useful as a transfection agent. The invention further describes transfection reagents, transfection particles containing this lipidoid, and their use.

    ##STR00001##

    Claims

    1. Lipidoids of general formula (I) ##STR00018## wherein X is selected from a group comprising C(?O)NH, C(?O)O, C(?S)O, C(?O)S, C(?S)S, C(?O)NHNH, CH.sub.2, O, OC(?O), S, SC(?O), NH, NHNH, NHC(?O), NHNHC(?O), C?C, CH?CH, a five-membered heterocycle containing at least 2 nitrogen atoms, CH.sub.2C(?O)NH, CH.sub.2C(?O)O, CH.sub.2C(?S)O, CH.sub.2C(?S)S, CH.sub.2C(?O)NHNH, N?CH, CH?N, NHN?CH, and CH?NNH; Y is selected from a group comprising alkylene C.sub.2-C.sub.10 chains; R.sup.1 are the same or different from each other, each R.sup.1 being independently selected from the group comprising alkyl C.sub.1-C.sub.46, alkenyl C.sub.2-C.sub.46, alkynyl C.sub.2-C.sub.46, and wherein said alkyl, alkenyl or alkynyl may be linear or branched, OC(?O), C(?O)O, SS, C(?O)NH, NHC(?O), O, and S; wherein if R.sup.1 is alkyl C.sub.1-C.sub.4, then one hydrogen from the terminal CH.sub.3 group may be substituted with Z; with the proviso that at least one R.sup.1 contains at least 8 carbon atoms; Z are the same or different from each other, each Z being independently selected from the group comprising hydrogen, OH, CH.sub.3, CH.sub.2OH, NH.sub.2, C(?O)NH.sub.2, CONH(CH.sub.2).sub.2OH, CON[(CH.sub.2).sub.2OH].sub.2, CONHCH(CH.sub.2OH).sub.2, CONHCH.sub.2CH(OH)CH.sub.2OH, CONH(CH.sub.2).sub.2C(?O)NH.sub.2, CON[CH.sub.2C(?O)NH.sub.2].sub.2, CONHCH[C(?O)NH.sub.2].sub.2, CONH(CH.sub.2).sub.2NHC(?O)NH.sub.2, CONH(CH.sub.2).sub.3N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.2SO.sub.3.sup.?, CONH(CH.sub.2).sub.3N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.3SO.sub.3.sup.?, CONH(CH.sub.2).sub.3N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.2COO.sup.?, COO(CH.sub.2).sub.2OP(?O)(O.sup.?)O(CH.sub.2).sub.2N.sup.+(CH.sub.3).sub.3, N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.3SO.sub.3.sup.?, N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.2COO.sup.?, ##STR00019## wherein R.sup.2 is independently selected from hydrogen and CH.sub.3; E is independently selected from O and S atoms; n is an integer within the range of from 1 to 5; and T are the same or different from each other, each T being independently selected from the group comprising XYN(R.sup.1).sub.2, C(?O)O(C.sub.1-C.sub.3 alkyl), C(?O)OCH.sub.2CH.sub.2OH, C(?O)NH.sub.2, ##STR00020## C(?O)OH, CONH(CH.sub.2).sub.2OH, CON[(CH.sub.2).sub.2OH].sub.2, CONHCH(CH.sub.2OH).sub.2, CONHCH.sub.2CH(OH)CH.sub.2OH, CONH(CH.sub.2).sub.2C(?O)NH.sub.2, CON[CH.sub.2C(?O)NH.sub.2].sub.2, CONHCH[C(?O)NH.sub.2].sub.2, CONH(CH.sub.2).sub.2NHC(?O)NH.sub.2, CONH(CH.sub.2).sub.3N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.2SO.sub.3.sup.?, CONH(CH.sub.2).sub.3N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.3SO.sub.3.sup.?, CONH(CH.sub.2).sub.3N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.2COO.sup.?, COO(CH.sub.2).sub.2OP(?O)(O)O(CH.sub.2).sub.2N.sup.+(CH.sub.3).sub.3, ##STR00021## OH, O(C.sub.1-C.sub.3 alkyl), NH.sub.2, NHC(?O)CH.sub.3, NHS(?O).sub.2CH.sub.3, NHC(?O)N(CH.sub.3).sub.2, NHC(?O)NH(CH.sub.3), NHC(?S)N(CH.sub.3).sub.2, NHC(?S)NH(CH.sub.3), NHC(?NCN)NH.sub.2, NHC(?NCN)NH(CH.sub.3), NHC(?NCN)N(CH.sub.3).sub.2, NHC[?NS(?O).sub.2NH.sub.2]NH.sub.2, N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.3SO.sub.3.sup.?, N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.2COO.sup.?, ##STR00022## wherein R.sup.2, E and n are as defined above; and/or if Z is OH or CH.sub.2OH, and T is C(?O)OH, then Z together with T and three carbon atoms between them may form a cyclic lactone containing 4 to 5 carbon atoms; and pharmaceutically acceptable salts, addition salts and solvates thereof.

    2. The lipidoid according to claim 1, wherein X is selected from a group consisting of C(?O)NH, C(?O)O, C(?O)NHNH, OC(?O), O, NHC(?O), NHNHC(?O), a five-membered heterocycle containing at least 2 nitrogen atoms.

    3. The lipidoid according to claim 1, wherein R.sup.1 are independently selected from the group consisting of alkyl C.sub.1-C.sub.46, and alkenyl C.sub.2-C.sub.46.

    4. The lipidoid according to claim 1, wherein all R.sup.1 in the molecule are the same or all nitrogen atoms in the molecule, bearing R.sup.1 groups, are substituted identically either by two identical R.sup.1 or by two different R.sup.1.

    5. The lipidoid according to claim 1, wherein Z is selected from the group consisting of hydrogen, OH, CH.sub.3, CH.sub.2OH, NH.sub.2, C(?O)NH.sub.2, CONH(CH.sub.2).sub.2OH, CON[(CH.sub.2).sub.2OH].sub.2, CONHCH(CH.sub.2OH).sub.2, CONHCH.sub.2CH(OH)CH.sub.2OH, CONH(CH.sub.2).sub.2C(?O)NH.sub.2, CON[CH.sub.2C(?O)NH.sub.2].sub.2, CONHCH[C(?O)NH.sub.2].sub.2, CONH(CH.sub.2).sub.2NHC(?O)NH.sub.2, N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.3SO.sub.3.sup.?, N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.2COO.sup.?, ##STR00023## wherein R.sup.2, E and n are as defined in claim 1.

    6. The lipidoid according to claim 1, wherein T is selected from the group consisting of XYN(R.sup.1).sub.2, C(?O)O(C.sub.1-C.sub.3 alkyl), C(?O)OCH.sub.2CH.sub.2OH, C(?O)NH.sub.2, ##STR00024## C(?O)OH, CONH(CH.sub.2).sub.2OH, CON[(CH.sub.2).sub.2OH].sub.2, CONHCH(CH.sub.2OH).sub.2, CONHCH.sub.2CH(OH)CH.sub.2OH, CONH(CH.sub.2).sub.2C(?O)NH.sub.2, CON[CH.sub.2C(?O)NH.sub.2].sub.2, CONHCH[C(?O)NH.sub.2].sub.2, CONH(CH.sub.2).sub.2NHC(?O)NH.sub.2, CONH(CH.sub.2).sub.3N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.2SO.sub.3.sup.?, CONH(CH.sub.2).sub.3N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.3SO.sub.3.sup.?, CONH(CH.sub.2).sub.3N.sup.+(CH.sub.3).sub.2(CH.sub.2).sub.2COO.sup.?, COO(CH.sub.2).sub.2OP(?O)(O)O(CH.sub.2).sub.2N.sup.+(CH.sub.3).sub.3, ##STR00025## wherein R.sup.2, E and n are as defined in claim 1.

    7. The lipidoid according to claim 1, wherein at least one substituent T is XYN(R.sup.1).

    8. The lipidoid according to claim 1, wherein Z is selected from the group consisting of hydrogen, OH, CH.sub.3, CH.sub.2OH; and T is selected from the group consisting of XYN(R.sup.1).sub.2, C(?O)O(C.sub.1-C.sub.3 alkyl), ##STR00026## C(?O)OH; and/or if Z is OH or CH.sub.2OH, and T is C(?O)OH, then Z together with T and three carbon atoms between them may form a cyclic lactone containing 4 to 5 carbon atoms.

    9. The lipidoid according to claim 1, wherein: X is C(?O)NH or NHC(?O); Y is selected from (CH.sub.2).sub.3, (CH.sub.2).sub.4, (CH.sub.2).sub.5, and (CH.sub.2).sub.6; R.sup.1 is selected from the group comprising linear or branched alkyl C.sub.10-C.sub.15; be and linear or branched alkenyl C.sub.12-C.sub.18; T is selected from XYN(R.sup.1).sub.2, C(?O)OCH.sub.3, ##STR00027## and C(?O)OH; Z is selected from H, CH.sub.3, and CH.sub.2OH.

    10. The lipidoid according to claim 1, wherein the lipidoid of general formula (I) contains at least two R.sup.1 substituents, each of them containing at least 8 carbon atoms.

    11. A transfection agent comprising at least one lipidoid of general formula (I) according to claim 1, and at least one helper lipid; wherein the helper lipid is selected from the group comprising cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethyleneglycol)-2000, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-poly(ethyleneglycol)-2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine poly(ethyleneglycol)-2000, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine poly(ethyleneglycol)-2000.

    12. The transfection agent according to claim 11, comprising at least one lipidoid of general formula (I) in an amount of from 10 to 50 mol. %, and at least one helper lipid in an amount of from 50 to 90 mol. %.

    13. The transfection agent according to claim 11, comprising at least one lipidoid of general formula (I) in an amount of from 15 to 40 mol. %, cholesterol in an amount of from 30 to 55 mol. %, and at least one helper lipid in an amount of from 20 to 50 mol. %; wherein the helper lipid is selected from the group comprising 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethyleneglycol)-2000, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-poly(ethyleneglycol)-2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine poly(ethyleneglycol)-2000, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine poly(ethyleneglycol)-2000.

    14. A transfection particle comprising at least one lipidoid of general formula (I) according to claim 1, at least one nucleic acid and/or a part thereof and/or nucleic acid derivative; and also at least one helper lipid.

    15. The lipidoid of general formula (I) according to claim 1 for in vitro transfection of cells or tissues with nucleic acid and/or a part thereof and/or nucleic acid derivative.

    16. The lipidoid of general formula (I) according to claim 15 for silencing or activating chromosomal gene(s), silencing or activating immunogenes, inhibiting or activating signaling pathways, editing genome or transcriptome, or enabling the expression of the protein(s) encoded by the nucleic acid.

    17. The lipidoid of general formula (I) according to claim 1 for transfection of cells or tissues with nucleic acid and/or a part thereof and/or nucleic acid derivative in vivo, excluding the transfection of human embryos for industrial or commercial purposes and excluding the modification of a human germ line.

    18. The lipidoid of general formula (I) according to claim 1 for silencing or activating chromosomal gene(s), silencing or activating immunogenes, inhibiting or activating signaling pathways, editing genome or transcriptome, or enabling the expression of the protein(s) encoded by the nucleic acid.

    19. The method of administering a medicament comprising the lipidoid of general formula (I) according claim 1.

    20. The lipidoid of general formula (I) according to claim 1 for a prophylactic vaccine.

    21. The lipidoid of general formula (I) according to claim 1 for cosmetic preparations in delivering the active ingredient to the site of action.

    Description

    BRIEF DESCRIPTION OF DRAWINGS

    [0129] FIG. 1. Synthetic scheme of lipidoids 3 and 7.

    [0130] FIG. 2. Synthetic scheme of lipidoids 8 and 11.

    [0131] FIG. 3. Synthetic scheme of lipidoid 15.

    [0132] FIG. 4. Synthetic scheme of lipidoids 19 and 23.

    [0133] FIG. 5. Synthetic scheme of lipidoids 27 and 31.

    [0134] FIG. 6. Synthetic scheme of lipidoids 32, 33, 35 and 36.

    [0135] FIG. 7. Synthetic scheme of precursors 39 and 44 and lipidoids 40-42.

    [0136] FIG. 8. Synthetic scheme of lipidoids 47, 48, 50 and 52.

    [0137] FIG. 9. Synthetic scheme of lipidoid 56.

    [0138] FIG. 10. Functional delivery of mRNA-LNP (B40) in murine liver in comparison to benchmark LNP (B45) analyzed by histology.

    [0139] FIG. 11. Functional delivery of mRNA-LNP (B40) in murine liver in comparison to benchmark LNP (B45) analyzed by PCR amplification ofthe genomic DNA. Lanes marked from 1 to 4 relate to B40. Lanes marked from 5 to 8 relate to B45.

    EXAMPLES

    List of Abbreviations

    [0140] eq. equivalent [0141] R.sub.f retention factor [0142] TLC thin-layer chromatography [0143] RVE rotary vacuum evaporator [0144] rt room temperature [0145] v/v volume/volume [0146] br s broad signal [0147] s singlet [0148] d doublet [0149] t triplet [0150] m multiplet [0151] dd doublet of doublets [0152] J interaction constant [0153] ? chemical shift [0154] HRMS high-resolution mass spectrometry [0155] EI electron ionization [0156] ESI electrospray ionization [0157] MALDI matrix-assisted laser desorption/ionisation [0158] GC-MS gas chromatographymass spectrometry [0159] IR infrared spectroscopy [0160] NMR nuclear magnetic resonance [0161] CE5 95:5 (v/v) cyclohexane-ethylacetate mixture [0162] CE20 80:20 (v/v) cyclohexane-ethylacetate mixture [0163] CE50 50:50 (v/v) cyclohexane-ethylacetate mixture [0164] D1 75:22:3 (v/v/v) dichloromethane-methanol-25% aqueous NH.sub.3 mixture [0165] D2 175:22:3 (v/v/v) dichloromethane-methanol-25% aqueous NH.sub.3 mixture [0166] D3 275:22:3 (v/v/v) dichloromethane-methanol-25% aqueous NH.sub.3 mixture [0167] D4 375:22:3 (v/v/v) dichloromethane-methanol-25% aqueous NH.sub.3 mixture [0168] TFA trifluoroacetic acid [0169] DIPEA N,N-diisopropylethylamine [0170] DMF N,N-dimethylformamide [0171] DCM dichloromethane [0172] ACN acetonitrile [0173] DIC diisopropylcarbodiimide [0174] DMAP 4-dimethylaminopyridine [0175] PyBroP bromotripyrrolidinophosphonium hexafluorophosphate [0176] TCE 1,1,2,2-tetrachloroethane [0177] LNP lipid nanoparticles [0178] NA nucleic acid [0179] DNA deoxyribonucleic acid [0180] RNA ribonucleic acid [0181] mRNA messenger RNA [0182] siRNA small interfering RNA [0183] tRNA transfer RNA [0184] miRNA micro RNA [0185] ssDNA/RNA single-stranded DNA/RNA [0186] dsDNA/RNA double-stranded DNA/RNA [0187] DMG-PEG.sub.2000 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 [0188] DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [0189] DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine [0190] DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine [0191] DOPE-Cy5 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5) [0192] Lip2000 Lipofectamine? 2000 (Invitrogen) [0193] h hour

    Example 1

    N.sup.1,N.sup.1-Didodecylethan-1,6-diamine 2

    [0194] A 500 ml round-bottom flask equipped with a calcium chloride drying tube and magnetic stirrer was filled with a solution of N.sup.1-terc-butyloxycarbonyl-1,6-diaminohexane (5.0 g, 23.11 mmol) in DCM (100 ml) and cooled to 0? C. in an ice bath. With intensive stirring, n-dodecylaldehyde (15.38 ml, 69.34 mmol, 3 eq.) was added, followed by sodium triacetoxyborohydride (14.70 g, 69.34 mmol, 3 eq.) in three portions over 10 minutes. The cooling bath was removed, and the reaction mixture was stirred at room temperature for 2 h.

    [0195] The progress of the reaction was monitored by TLC using an 80:20 (v/v) hexane-ethylacetate mobile phase on a TLC plate pre-saturated with ammonia (detection with ninhydrin). After completion of the reaction, aqueous NaOH solution (1 M, 200 ml) was added, the reaction mixture was stirred for 15 min, then poured into a separatory funnel and diluted with water (300 ml). The product was extracted with DCM (300 ml, 2?50 ml), the combined organic phase was washed with brine (50 ml), dried over anhydrous sodium sulphate, filtered through an S2 frit, and the solvents were evaporated in an RVE. The dark oily residue was purified by silica gel column chromatography using a linear gradient of ethyl acetate in hexane (10-30%). Amine 1 (3.67 g, 28.7%) was obtained as a yellowish oil.

    [0196] Trifluoroacetic acid (10 ml) was added to a solution of compound 1 in DCM (10 ml), cooled to 0? C. with stirring in an ice bath, and the reaction mixture was left at 0? C. for 3 h. The solution was then poured into a 1 1 separatory flask, diluted with 20% aqueous Na.sub.2CO.sub.3 (300 ml), and the product was extracted with DCM (250 ml, 2?50 ml). The combined organic phase was washed with brine (100 ml), dried over anhydrous sodium sulphate, filtered through an S2 frit, and the solvents were evaporated in an RVE. The crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (0-70%). The diamine 2 (2.17 g, 72.2% yield; R.sub.f 0.31 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained in the form of a yellowish oil.

    [0197] .sup.1H NMR (600 MHz, CDCl.sub.3): ?=2.73, 2.65, 2.57, 1.56, 1.52, 1.51, 1.36, 1.31, 1.28, 1.25-1.29, 1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=53.51, 53.20, 41.62, 32.40, 31.89, 29.63, 29.61, 29.57, 29.44, 29.32, 27.39, 27.09, 26.53, 25.65, 25.02, 22.66, 14.10 ppm. IR (film): v.sub.max/cm.sup.?1=3374 w and 3294 w (v NH.sub.2), 2797 m (v.sub.s NCH.sub.2), 2956 s (v.sub.as CH.sub.3), 2924 vs (v.sub.as CH.sub.2), 2853 s (v.sub.s CH.sub.2), 1467 m and 1455 m, sh (p, CH.sub.2 and ?.sub.as CH.sub.3), 1378 w and 1367 w (?.sub.s CH.sub.3), 721 m (?.sub.as CH.sub.2). HRMS (ESI): m/z calculated for C.sub.30H.sub.65N.sub.2 [M+H].sup.+ 453.51423; found 453.51340.

    cis,cis-N.sup.1,N.sup.3,N.sup.5-Tris(6-(didodecylamino)hexyl)cyclohexane-1,3,5-tricarboxamide 3

    [0198] DMF (2 ?l) andthionylchloride (300 ?l) were addedto cis,cis-1,3,5-cyclohexanetricarboxylic acid (17 mg, 0.079 mmol) and the suspension was stirred for 30 min at 70? C. in a sealed vial, gradually clarifying the reaction mixture to a homogeneous colorless solution. Excess SOCl.sub.2 was blown off with a stream of dry nitrogen at 70? C., the residue was dried under vacuum (10 min), and cooled to rt. A solution of N.sup.1,N.sup.1-didodecylhexane-1,6-diamine 2 (142 mg, 0.315 mmol, 4 eq.) and DIPEA (137 ?l, 0.786 mmol, 10 eq.) in a mixture of DCM (1.5 ml) and DMF (0.5 ml), was added via septum using a syringe and the reaction mixture was stirred for 10 min. Subsequently, the reaction mixture was sorbed onto chromatographic silica gel (10 g), and the solvents were removed in an RVE. The residue was purified by silicagel column chromatography (40 g) using a linear gradient of D1 in DCM (0-55%). Lipidoid 3 was obtained (81 mg, 67.7% yield; R.sub.f 0.36 in mobile phase D2, detection with ninhydrin) in the form of a yellowish semi-solid.

    [0199] .sup.1H NMR (600 MHz, CDCl.sub.3): ?=7.80, 3.20, 3.02-2.96, 2.60, 2.07, 1.81-1.74, 1.37, 1.325, 1.28-1.23, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=175.90, 52.85, 52.22, 42.83, 39.42, 31.87, 31.66, 29.58, 29.48, 29.43, 29.30, 29.08, 26.84, 26.18, 26.00, 23.50, 23.16, 22.65, 14.09 ppm. IR (CCl.sub.4): v.sub.max/cm.sup.?1=3293 m (v NH), 1640 s (amide I) and 1550 m (amide II), 2964 s, sh (v.sub.as CH.sub.3), 2871 m, sh (v.sub.s CH.sub.3), 2927 vs (v.sub.as CH.sub.2), 2856 s, sh (v.sub.s CH.sub.2), 1468 m and 1460 m, 1445 w (?.sub.s CH.sub.2 and ?.sub.as CH.sub.3), 1378 w (?.sub.s CH.sub.3), 721 w (fas CH.sub.2 and ?.sub.as CH.sub.2); HRMS (MALDI): m/z calculated for C.sub.99H.sub.199N.sub.6O.sub.3 [M+H].sup.+ 1520.5598; found 1520.5598.

    Example 2

    [0200] N.sup.1,N.sup.1-Di((hexyloxycarbonyl)butyl)hexane-1,6-diamine 6 1-hexanol (2.48 ml, 19.89 mmol, 1.2 eq), DMAP (61 mg, 0.50 mmol, 0.03 eq.) and DIC (3.37 ml, 21.54 mmol, 1.3 eq.) were added to a solution of 5-bromopentanoic acid (3.00 g, 16.57 mmol) in DCM (60 ml) and the reaction mixture was stirred for 1 h at rt. Subsequently, the reaction mixture was sorbed onto chromatographic silica gel (16 g), the solvents were removed in an RVE, and the residue was purified by silicagel column chromatography (80 g) using a linear gradient of ethyl acetate in cyclohexane (0-10%).

    [0201] Hexyl 5-bromopentanoate 4 (3.938 g, 89.6% yield; R.sub.f 0.31 in mobile phase CE5, detection KMnO.sub.4) was obtained as a colourless liquid.

    [0202] Hexyl 5-bromopentanoate 4 (1.53 g, 5.78 mmol, 2.5 eq.) and anhydrous K.sub.2CO.sub.3 (3.19 g, 23.11 mmol, 10 eq.) were added to a solution of N-tert-butyloxycarbonyl-1,6-diaminohexane (0.50 g, 2.31 mmol) in anhydrous ACN (10 ml) and the reaction mixture was stirred vigorously at 35? C. for 2 days. Subsequently, the reaction mixture was sorbed onto chromatographic silicagel (16 g), the solvents were removed in an RVE, and the residue was purified by silicagel column chromatography (40 g) using a linear gradient of ethyl acetate in cyclohexane (0-100%). Amine 5 was obtained (1.080 g, 79.9% yield; R.sub.f 0.31 in mobile phase CE50 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) in the form of slightly yellowish oil.

    [0203] Trifluoroacetic acid (4 ml) was added to a solution of compound 5 in DCM (4 ml) cooled to 0? C. with stirring in an ice bath, and the reaction mixture was left at 0? C. for 1 h. The solution was then poured into a 500 ml separatory flask, diluted with 20% aqueous NaHCO.sub.3 (200 ml), and the product was extracted with DCM (150 ml, 2?50 ml). The combined organic phase was washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered through an S2 frit, sorbed onto chromatographic silicagel (16 g), the solvents removed in an RVE, and the residue was purified by silicagel column chromatography (40 g) using a linear gradient of D1 in DCM (0-80%). Diamine 6 was obtained (0.788 g, 86.9% yield; R.sub.f 0.14 in mobile phase D2, detection with ninhydrin) as a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=4.05, 3.63, 3.21, 3.15, 2.79, 2.68, 2.60, 2.51, 2.42, 2.32, 1.61, 1.50, 1.36-1.28, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=173.62, 64.52, 53.68, 53.29, 41.22, 40.26, 34.03, 31.41, 28.58, 25.75, 25.57, 22.80, 22.51, 13.98 ppm.

    [0204] HRMS (ESI): m/z calculated for C.sub.28H.sub.57O.sub.4N.sub.2 [M+H].sup.+ 485.43128; found 485.43052.

    cis,cis-N.sup.1,N.sup.3,N-Tris(6-(di((hexyloxycarbonyl)butyl)amino)hexyl)cyclohexane-1,3,5-tricarboxamide 7

    [0205] Lipidoid 7 was prepared from cis,cis-1,3,5-cyclohexanetricarboxylic acid (21 mg, 0.097 mmol), N,N-di((hexyloxycarbonyl) butyl)hexane-1,6-diamine 6 (188 mg, 389 mmol, 4 eq.) and DIPEA (169 ?l, 0.971 mmol, 10 eq.) according to the procedure described for lipidoid 3 in Example 1. Lipidoid 7 (55 mg, 35%; R.sub.f 0.42 in mobile phase D2, detection with ninhydrin) was obtained as a yellowish semi-solid. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=6.23, 4.05, 3.205, 2.65, 2.34, 2.30, 2.085, 1.63-1.60, 1.49, 1.33-1.29, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=174.35, 173.37, 64.60, 53.35, 52.88, 43.98, 39.12, 33.79, 31.94, 31.40, 29.21, 28.57, 26.62, 26.33, 25.56, 22.58, 22.51, 13.98 ppm. IR (CCl.sub.4): v.sub.max/cm.sup.?1=1736 vs (v.sub.s C?O), 1173 m, 1075 w (v.sub.s CO), 3293 m (v NH), 1640 s (amide I) and 1551 m (amide II), 2955 s (v.sub.as CH.sub.3), 2933 vs (v.sub.asCH.sub.2), 2875 m, sh (v.sub.s CH.sub.3), 2860 m (v.sub.s CH.sub.2), 1467 m and 1460 m (0, CH.sub.2 and ?.sub.as CH.sub.3), 1379 w (?.sub.s CH.sub.3), 724 w (?.sub.as CH.sub.2 and ?.sub.as CH.sub.2). HRMS (MALDI): m/z calculated for C.sub.93H.sub.175N.sub.6O.sub.15[M+H].sup.+ 1616.3110; found 1616.3095.

    Example 3

    cis,cis-N.sup.1,N.sup.3,N.sup.5-Tris(6-(didodecylamino)hexyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide 8

    [0206] Lipidoid 8 was prepared from cis,cis-1,3,5-trimethyl-1,3,5-cyclohexanetricarboxylic acid (20 mg, 0.077 mmol), N,N-di(dodecyl)hexane-1,6-diamine 2 (140 mg, 310 mmol, 4 eq.) and DIPEA (135 ?l, 0.774 mmol, 10 eq.) according to the procedure described for lipidoid 3 in Example 1. Lipidoid 8 (64 mg, 53%; R.sub.f0.43 in mobile phase D2, detection with ninhydrin) was obtained in the form of a slightly yellowish solid oil. IR (CCl.sub.4): v.sub.max/cm.sup.?1=3288 w, br (v NH), 1651 m (amide I), 1585 w, br, sh and 1559 m, br (amide II), 2956 s (v.sub.as CH.sub.3), 2927 vs (v.sub.as CH.sub.2), 2878 m, sh (v, CH.sub.3), 2855 s (v, CH.sub.2), 1468 m, and 1459 m, sh, 1448 m, sh (?.sub.s CH.sub.2 and ?.sub.as CH.sub.3), 1378 w (?.sub.s CH.sub.3); 721 w (?.sub.as CH.sub.2 and ?.sub.as CH.sub.2). HRMS (MALDI): m/z calculated for C.sub.102H.sub.205N.sub.6O.sub.3 [M+H].sup.+ 1562.6068; found 1562.6011.

    Example 4

    N.sup.1,N.sup.1-Didodecylpropane-1,3-diamine 10

    [0207] Amine 9 was prepared from N.sup.1-tert-butyloxycarbonyl-1,3-diaminopropane (6.0 g, 34.43 mmol), n-dodecylaldehyde (22.91 ml, 103.30 mmol, 3 eq.) and sodium triacetoxyborohydride (21.89 g, 103.3 mmol, 3 eq.) according to the procedure described for compound 1 in Example 1. Amine 9 was obtained as a yellowish oil (7.72 g, 43.9%).

    [0208] The deprotection of amine 9 was performed according to the procedure described for compound 2 in Example 1; diamine 10 (4.26 g, 68.6%; R.sub.f 0.35 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained in the form of a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=3.07, 2.70, 2.50, 1.81, 1.46, 1.28, 1.26, 1.25-1.29, 1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=53.70, 53.30, 41.18, 31.90, 29.64, 29.62, 29.60, 29.58, 29.48, 29.33, 27.42, 25.71, 23.87, 22.67, 14.10 ppm. IR (film): v.sub.max/cm.sup.?1=3361 w and 3274 w (v NH.sub.2), 2803 m (v.sub.s NCH.sub.2), 2954 s (v.sub.a CH.sub.3), 2924 vs (v.sub.as CH.sub.2), 2853 s (v.sub.s CH.sub.2), 1467 m and 1456 m, sh (p, CH.sub.2 and ?.sub.as CH.sub.3), 1378 w and 1364 w (?.sub.s CH.sub.3), 720 m (P.sub.as CH.sub.2). HRMS (ESI): m/z calculated for C.sub.27H.sub.59N.sub.2[M+H].sup.+ 411.46728; found 411.46652.

    cis,cis-N.sup.1,N.sup.3,N.sup.5-Tris(3-(didodecylamino)propyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide 11

    [0209] Lipidoid 11 was prepared from cis,cis-1,3,5-trimethyl-1,3,5-cyclohexanetricarboxylic acid (20 mg, 0.077 mmol), N,N-di(dodecyl)propane-1,3-diamine 10 (127 mg, 310 mmol, 4 eq.) and DIPEA (135 ?l, 0.774 mmol, 10 eq.) according to the procedure described for lipidoid 3 in Example 1. Lipidoid 11 (41 mg, 37% yield; R.sub.f0.36 in mobile phase D2, detection with ninhydrin) was obtained in the form of a slightly yellowish solid oil. IR (CCl.sub.4): v.sub.max/cm.sup.?1=3303 w, vbr (v NH), 1679 s, sh (amide I), 1513 w, br, sh (amide II), 2956 s (v.sub.as CH.sub.3), 2927 vs (v.sub.as CH.sub.2), 2878 m, sh (v.sub.s CH.sub.3), 2855 s (v.sub.s CH.sub.2), 1464 m, sh (p, CH.sub.2 and 6.sub.as CH.sub.3), 1380 w (?.sub.s CH.sub.3); 721 w (?.sub.as CH.sub.2 and ?.sub.as CH.sub.2). HRMS (MALDI): m/z calculated for C.sub.93H.sub.187N.sub.6O.sub.3 [M+H].sup.+ 1436.4665; found 1436.4629.

    Example 5

    Linoleylaldehyde 12

    [0210] Dess-Martin periodinane (4.45 g, 10.49 mmol, 1.3 eq.) was added to a solution of linoleylalcohol (2.50 ml, 8.07 mmol) in DCM (120 ml) cooled to 0? C. with an ice bath and the mixture was stirred at 0? C. for 4 h. The reaction was then quenched by the addition of sodium thiosulfate solution (20 g Na.sub.2S.sub.2O.sub.3, 5H.sub.2O/100 ml H.sub.2O) and saturated aqueous sodium bicarbonate solution (50 ml), and stirred at rt for 1 h until the initially milky solution turned clear. The solution was poured into a 1000 ml separatory flask, diluted with water (150 ml), and the product was extracted with DCM (150 ml, 2?50 ml). The combined organic phase was washed with brine (150 ml), dried over anhydrous sodium sulfate, filtered through an S2 frit, and the solvents were evaporated on a RVE. The crude product was purified by silicagel column chromatography (isocratic conditions, 5% ethyl acetate in cyclohexane). Aldehyde 12 (1.271 g, 59.6% yield; R.sub.f 0.36 in mobile phase CE5, detection with KMnO.sub.4) was obtained as a colorless oil.

    N.sup.1,N.sup.1-Di((9Z,12Z)-octadeca-9,12-dien-1-yl)hexane-1,6-diamine 14

    [0211] Amine 13 was prepared from N.sup.1-tert-butyloxycarbonyl-1,6-diaminohexane (0.345 g, 1.59 mmol), aldehyde 12 (1.27 g, 4.78 mmol, 3 eq.) and sodium triacetoxyborohydride (1.01 g, 4.78 mmol, 3 eq.) according to the procedure described for compound 1 in Example 1. Amine 13 was obtained as a yellowish oil (1.08 g, 94.9% yield; R.sub.f 0.18 in CE20 mobile phase, detection with ninhydrin).

    [0212] Deprotection of amine 13 was performed in a mixture of TFA (4 ml) and DCM (5 ml) according to the procedure described for compound 2 in Example 1; diamine 14 (0.594 g, 64.0%; R.sub.f 0.13 in mobile phase D2, detection with ninhydrin) was obtained as a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=5.30-5.40, 2.765, 2.73, 2.67, 2.59, 2.04, 1.51, 1.385, 1.37, 1.34, 1.295, 1.29, 1.28-1.34, 1.28, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=130.19, 130.06, 127.99, 127.89, 53.49, 53.45, 41.67, 32.52, 31.50, 29.62, 29.46, 29.20-29.48, 27.37, 27.20, 27.18, 26.52, 25.61, 22.56, 14.06 ppm. IR (CCl.sub.4): v.sub.max/cm.sup.?1=3011 s (v.sub.as=CH); 1646-1673 m (v C?C); 3455 w (v.sub.as NH.sub.2); 3394 (v.sub.s NH.sub.2); 1620 w (0, NH.sub.2); 1087 m (v CNH.sub.2); 2957 s, sh (v.sub.as CH.sub.3); 2928 vs (v.sub.as CH.sub.2); 2873 s, sh (v.sub.s CH.sub.3); 2856 vs (v.sub.s CH.sub.2); 2801 m (v.sub.s NCH.sub.2); 1467 m and 1457 m, sh (p, CH.sub.2 and ?.sub.as CH.sub.3); 1378 m (?.sub.s CH.sub.3); 721 m (?.sub.as and ?.sub.as CH.sub.2). HRMS: m/z calculated for C.sub.42H.sub.81N.sub.2 [M+H].sup.+ 613.63943; found 613.63899.

    cis,cis-N.sup.1,N.sup.3,N-Tris(6-(di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)hexyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide 15

    [0213] Lipidoid 15 was prepared from cis,cis-1,3,5-trimethyl-1,3,5-cyclohexanetricarboxylic acid (17 mg, 0.066 mmol), N.sup.1,N-di((9Z,12Z)-octadeca-9,12-dien-1-yl)hexane-1,6-diamine 14 (161 mg, 273 mmol, 4 eq.) and DIPEA (115 ?l, 0.658 mmol, 10 eq.) according to the procedure described for lipidoid 3 in Example 1.

    [0214] Lipidoid 15 (86 mg, 64% yield; R.sub.f 0.36 in mobile phase D2, detection with ninhydrin) was obtained as a pale yellow solid. IR (CCl.sub.4): v.sub.max/cm.sup.?1=3348 w, br, sh and 3302 w, br (v NH), 1656 m and 1635 m, sh (amide I+v C?C), 1565 w, sh and 1557 w, br (amide II), 3011 m (v.sub.as=CH), 2956 s (v.sub.as CH.sub.3), 2929 vs (v.sub.aCH.sub.2), 2875 m, sh (v.sub.s CH.sub.3), 2856 s (v.sub.s CH.sub.2), 1467 m and 1450 m, sh, (p, CH.sub.2 and ?.sub.as CH.sub.3), 1379 w (?.sub.s CH.sub.3); 720 w (?.sub.as CH.sub.2+?.sub.as CH.sub.2+?=CH). HRMS (MALDI): m/z calculated for C.sub.138H.sub.253N.sub.6O.sub.3 [M+H].sup.+ 2042.9829; found 2042.9861.

    Example 6

    Compound 18

    [0215] DIC (4.41 mL, 28.7 mmol, 1.6 eq.) and DMAP (88 mg, 0.72 mmol, 0.04 eq.) were added to a solution of 6-bromohexanoic acid (3.50 g, 17.9 mmol) and octan-2-ol (3.51 g, 26.9 mmol, 1.5 eq.) in DCM (30 mL), and the mixture was stirred overnight at rt. The reaction mixture was then adsorbed onto silica (16 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (80 g, elution with a linear gradient of ethyl acetate in cyclohexane, 0-10%) to yield the compound 16 (4.02 g, 77%; R.sub.f 0.41 in CE5, visualization by KMnO.sub.4) as a colorless oil.

    [0216] Bromoester 16 (4.00 g, 13.1 mmol, 2.6 eq.) and potassium carbonate (7.23 g, 52.3 mmol, 10 eq.) were added to a solution of N-Boc-1,6-hexanediamine (1.13 g, 5.23 mmol, 1 eq.) in anhydrous ACN (10 mL), and the mixture was stirred at 40? C. for 3 days. The reaction mixture was then adsorbed onto silica (16 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (80 g, elution with a linear gradient of ethyl acetate in cyclohexane, 0-100%) to yield the compound 17 (2.80 g, 80%; R.sub.f 0.58 in CE50 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as a pale-yellow oil.

    [0217] Hydrochloric acid in dioxane (4 mL, 4 M) was added to a solution of compound 17 (2.75 g, 4.11 mmol) in anhydrous DCM (4 mL), and the reaction mixture was stirred at rt for 1 h. The solution was then poured into a 500 mL separatory funnel, diluted with saturated aqueous NaHCO.sub.3 (100 mL), and the product was extracted with diethyl ether (100 mL, 2?50 mL). The combined organic phases were washed with brine (75 mL), dried over anhydrous sodium sulfate, filtered through an S2 sintered glass, and the solvents were evaporated in an RVE. The crude product was purified by column chromatography on silica using a linear gradient of D1 in DCM (0-70%). The amine 18 (1.74 g, 74%; R.sub.f 0.25 in D2 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) was obtained as a pale-yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=4.90-4.77 (m), 2.72-2.62 (m), 2.39-2.29 (m), 2.21 (t, J=7.5 Hz), 1.64-1.44 (m), 1.47-1.32 (m), 1.30-1.17 (m), 1.15-1.08 (m), 0.86-0.78 (m) ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=173.42, 70.83, 53.89, 41.87, 35.98, 31.77, 29.13, 27.41, 27.17, 26.84, 26.69, 25.40, 25.09, 22.60, 20.04, 14.09 ppm. HRMS (ESI): m/z calculated for C.sub.34H.sub.68N.sub.2O.sub.4[M+H].sup.+ 569.5252; found 569.5247.

    Hexa(octan-2-yl) cis,cis-6,6,6,6,6,6-((((cyclohexane-1,3,5-tricar-bonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azanetriyl))hexahexanoate 19

    [0218] Thionylchloride (700 ?L) and DMF (6 ?L) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (80 mg, 370 ?mol), and the suspension was stirred for 3 h at 70? C. in a closed vial; during this time the suspension turned into a clear solution. Excess SOCl.sub.2 was removed at 70? C. with a stream of dry nitrogen, the residue was dried at rt in vacuo (20 min), and the obtained solid was dissolved under argon in anhydrous TCE (2.5 mL) and DIPEA (645 ?L, 3.70 mmol). Then, a solution of amine 18 (1.05 g, 1.85 mmol, 5 eq.) in anhydrous TCE (2.5 mL) was added and the reaction mixture was stirred for 40 min at rt. The reaction mixture was then evaporated in vacuo, redisolved in DCM, adsorbed onto silica (10 g), and the DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 5-35%) to yield the target compound 19 (489 mg, 71%) as a yellow waxy semi-solid. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=5.75, 4.88, 3.21, 2.42, 2.27, 2.22, 2.11, 1.62, 1.58, 1.56, 1.47, 1.46, 1.30, 1.28, 1.27, 1.26, 1.19, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): 173.88, 173.39, 70.82, 53.87, 53.77, 44.10, 39.44, 35.93, 34.67, 31.86, 31.73, 29.51, 29.09, 27.08, 26.74, 25.36, 25.01, 22.56, 20.01, 14.06 ppm.

    [0219] HRMS (MALDI): m/z calcd. for C.sub.111H.sub.211N.sub.6O.sub.15 [M+H].sup.+ 1868.5927; found 1868.5906.

    Example 7

    Compound 22

    [0220] DIC (3.60 mL, 23.0 mmol, 1.6 eq.) and DMAP (70 mg, 0.58 mmol, 0.04 eq.) were added to a solution of 7-bromoheptanoic acid (3.00 g, 14.4 mmol) and 3-methylhexan-1-ol (2.50 g, 21.5 mmol, 1.5 eq.) in DCM (30 mL), and the mixture was stirred overnight at rt. The reaction mixture was then adsorbed onto silica (16 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (80 g, elution with a linear gradient of ethyl acetate in cyclohexane, 0-10%) to yield the compound 20 (3.64 g, 83%; R.sub.f 0.27 in CE5, visualization by KMnO.sub.4) as a colorless oil.

    [0221] Bromoester 20 (3.63 g, 11.8 mmol, 2.6 eq.) and potassium carbonate (6.29 g, 45.5 mmol, 10 eq.) were added to a solution of N-Boc-hexane-1,6-diamine (0.98 g, 4.55 mmol, 1 eq.) in anhydrous ACN (10 mL), and the mixture was stirred at 40? C. for 3 days. The reaction mixture was then adsorbed onto silica (16 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (80 g, elution with a linear gradient of ethyl acetate in cyclohexane, 0-100%) to yield the compound 21 (2.90 g, 95%; R.sub.f 0.54 in CE50 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as a pale-yellow oil.

    [0222] Hydrochloric acid in dioxane (4 mL, 4 M) was added to a solution of compound 21 (2.85 g, 4.26 mmol) in anhydrous DCM (4 mL), and the reaction mixture was stirred at rt for 1 h. The solution was then poured into a 500 mL separatory funnel, diluted with saturated aqueous NaHCO.sub.3 (100 mL), and the product was extracted with diethyl ether (100 mL, 2?50 mL). The combined organic phases were washed with brine (75 mL), dried over anhydrous sodium sulfate, filtered through an S2 sintered glass, and the solvents were evaporated in an RVE. The crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (0-70%). The amine 22 (2.38 g, 96%; R.sub.f 0.48 in D2 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) was obtained as a pale-yellow oil.

    [0223] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=4.08-3.96 (m), 2.63-2.58 (m), 2.33-2.26 (m), 2.21 (t, J=7.5 Hz), 1.61-1.50 (m), 1.49-1.40 (m), 1.40-1.30 (m), 1.30-1.14 (m), 1.14-1.00 (m), 0.85-0.76 (m) ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): 173.85, 62.77, 54.13, 42.19, 38.98, 35.55, 34.35, 33.79, 29.56, 29.15, 27.49, 27.29, 26.88, 25.01, 19.95, 19.48, 14.26 ppm. HRMS (ESI): m/z calculated for C.sub.34H.sub.68N.sub.2O.sub.4[M+H].sup.+ 569.5252; found 569.5250.

    Hexakis(3-methylhexyl) cis,cis-7,7,7,7,7 ,7-((((cyclohexane-1,3,5-tricarbonyl) tris(azanediyl))tris(hexane-6,1-diyl))tris(azanetriyl))hexaheptanoate 23

    [0224] Thionylchloride (500 ?L) and DMF (4 ?L) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (60 mg, 278 ?mol), and the suspension was stirred for 3 h at 70? C. in a closed vial; during this time the suspension turned into a clear solution. Excess SOCl.sub.2 was removed at 70? C. with a stream of dry nitrogen, the residue was dried at rt in vacuo (20 min), and the obtained solid was dissolved under argon in anhydrous TCE (2 mL) and DIPEA (483 ?L, 2.78 mmol, 10 eq.). Then, a solution of amine 22 (632 mg, 1.11 mmol, 4 eq.) in anhydrous TCE (2 mL) was added and the reaction mixture was stirred for 40 min at rt. The reaction mixture was then evaporated in vacuo, redisolved in DCM, adsorbed onto silica (10 g), and the DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 0-46%) to yield the target compound 23 (346 mg, 67%; R.sub.f 0.66 in D2 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as a yellow waxy semi-solid. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=5.75, 4.09, 3.21, 2.41, 2.28, 2.22, 2.11, 1.64, 1.62, 1.61, 1.58, 1.54, 1.48, 1.44, 1.42, 1.33, 1.32, 1.29, 1.28, 1.13, 0.89, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=173.92, 173.87, 62.64, 53.90, 44.10, 39.45, 39.14, 35.51, 34.23, 31.86, 29.54, 29.52, 29.09, 27.22, 27.13, 26.77, 24.97, 19.94, 19.47, 14.26 ppm. HRMS (MALDI): m/z calcd. for C.sub.111H.sub.211N.sub.6O.sub.15 [M+H].sup.+ 1868.5927; found 1868.5905.

    Example 8

    Compound 24

    [0225] N,N-Diisopropylcarbodiimide (4.07 mL, 24.9 mmol, 1.6 eq.) was added to a solution containing 6-bromo-pentanoic acid (3.00 g, 16.6 mmol), DMAP (81 mg, 0.66 mmol, 0.04 eq.), and geraniol (4.36 mL, 24.9 mmol, 1.5 eq.) in anhydrous DCM (150 mL). The mixture was stirred overnight at rt. The reaction mixture was then adsorbed onto silica (20 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (120 g, elution with a linear gradient of ethyl acetate in cyclohexane, 0-10%) to yield the target compound 24 (4.80 g, 91%; R.sub.f 0.39 in CE5, visualization by KMnO.sub.4) as a pale-yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3) ? 5.37-5.30 (m, 1H), 5.11-5.05 (m, 1H), 4.60 (d, J=7.1 Hz, 2H), 3.41 (t, J=6.6 Hz, 2H), 2.35 (t, J=7.2 Hz, 2H), 2.15-2.01 (m, 4H), 1.95-1.86 (m, 2H), 1.83-1.74 (m, 2H), 1.70 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H).

    Compound 26

    [0226] Bromoester 24 (4.80 g, 15.1 mmol, 2.3 eq.) and potassium carbonate (7.27 g, 52.3 mmol, 8 eq.) were added to a solution of N-Boc-1,6-hexanediamine (1.42 g, 6.58 mmol, 1 eq.) in anhydrous ACN (30 mL), and the mixture was stirred at 45? C. for 24 h. The reaction mixture was then adsorbed onto silica (20 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (120 g, column pretreated with gaseous NH.sub.3, elution with a linear gradient of ethyl acetate in cyclohexane, 0-100%) to yield the compound 25 (2.80 g, 54%; R.sub.f 0.58 in CE50 on an NH.sub.3-pretreated TLC plate, visualization by KMnO.sub.4) as a pale-yellow oil.

    [0227] A flask equipped with a magnetic stir bar was loaded with compound 25 (2.80 g, 4.06 mmol), closed with a rubber septum and flushed with argon. ACN (10 mL) and DCM (20 mL) were added through the septum using a needle and syringe, the stirring was turned on and a solution of 4-toluenesulfonic acid monohydrate (2.32 g, 12.2 mmol, 3 eq.) in ACN (20 mL) was added at rt through the septum over the course of 5 min using a needle and syringe. The reaction mixture was stirred at rt for 6 h and was subsequently neutralized using a stream of gaseous ammonia. The reaction mixture was then adsorbed onto silica (20 g) and the crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (10-43%). The amine 26 (810 mg, 34%; R.sub.f 0.45 in D2 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) was obtained as a yellow oil. HRMS (ESI): m/z calcd. for C.sub.36H.sub.64N.sub.2O.sub.4 [M+H].sup.+ 589.4939; found 589.4936.

    Hexakis((E)-3,7-dimethylocta-2,6-dien-1-yl) cis,cis-5,5,5,5,5 ,5-((((cyclohexane-1,3,5-tricar-bonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azanetriyl))hexapentanoate 27

    [0228] Thionylchloride (700 ?L) and DMF (6 ?L) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (45 mg, 208 ?mol), and the suspension was stirred for 3 h at 70? C. in a closed vial; during this time the suspension turned into a clear solution. Excess SOCl.sub.2 was removed at 70? C. with a stream of dry nitrogen, the residue was dried at rt in vacuo (20 min), and the obtained solid was dissolved under argon in anhydrous TCE (1.5 mL) and DIPEA (363 ?L, 2.08 mmol, 10 eq.). Then, a solution of amine 26 (490 mg, 833 ?mol, 4 eq.) in anhydrous TCE (1.0 mL) was added and the reaction mixture was stirred for 40 min at rt. The reaction mixture was then evaporated in vacuo, redisolved in DCM, adsorbed onto silica (10 g), and the DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 0-31%) to yield the target compound 27 (262 mg, 65%; R.sub.f 0.59 in D2 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as a yellow waxy semi-solid. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=5.72, 5.32, 5.08, 4.58, 3.21, 2.44, 2.41, 2.32, 2.22, 2.11, 2.10, 2.09, 2.03, 1.69, 1.68, 1.61, 1.60, 1.59, 1.58, 1.47, 1.45, 1.29 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=173.85, 173.60, 142.16, 131.80, 123.72, 118.32, 62.25, 53.79, 53.45, 44.12, 39.52, 39.42, 34.14, 31.85, 29.48, 27.03, 26.72, 26.29, 25.67, 22.90, 17.68, 16.46 ppm. HRMS (MALDI): m/z calcd. For C.sub.117H1.sub.99N.sub.6O.sub.15 [M+H].sup.+ 1928.4988; found 1928.4967.

    Example 9

    Compound 28

    [0229] N,N-Diisopropylcarbodiimide (4.07 mL, 24.9 mmol, 1.6 eq.) was added to a solution containing 6-bromo-pentanoic acid (3.00 g, 16.6 mmol), DMAP (81 mg, 0.66 mmol, 0.04 eq.), and nerol (4.38 mL, 24.9 mmol, 1.5 eq.) in anhydrous DCM (60 mL). The mixture was stirred overnight at rt. The reaction mixture was then adsorbed onto silica (20 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (120 g, elution with a linear gradient of ethyl acetate in cyclohexane, 0-10%) to yield the target compound 28 (4.75 g, 90%; R.sub.f 0.39 in CE5, visualization by KMnO.sub.4) as a pale-yellow oil. .sup.1H NMR (401 MHz, CDCl.sub.3) ? 5.35 (t, J=6.5 Hz, 1H), 5.12-5.06 (m, 1H), 4.57 (dd, J=7.2, 1.1 Hz, 2H), 3.40 (t, J=6.6 Hz, 2H), 2.34 (t, J=7.3 Hz, 2H), 2.15-2.03 (m, 4H), 1.94-1.86 (m, 2H), 1.82-1.74 (m, 5H), 1.68 (d, J=1.4 Hz, 3H), 1.60 (s, 3H).

    Compound 30

    [0230] Bromoester 28 (4.75 g, 15.0 mmol, 2.3 eq.) and potassium carbonate (7.20 g, 52.1 mmol, 8 eq.) were added to a solution of N-Boc-1,6-hexanediamine (1.41 g, 6.51 mmol, 1 eq.) in anhydrous ACN (45 mL), and the mixture was stirred at 48? C. for 24 h. The reaction mixture was then adsorbed onto silica (20 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (120 g, column pretreated with gaseous NH.sub.3, elution with a linear gradient of ethyl acetate in cyclohexane, 0-100%) to yield the compound 29 (3.62 g, 81%; R.sub.f 0.54 in CE50 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as a pale-yellow oil.

    [0231] A flask equipped with a magnetic stir bar was loaded with compound 29 (3.62 g, 5.25 mmol), closed with a rubber septum and flushed with argon. DCM (10 mL) was added through the septum using a needle and syringe, the stirring was turned on and a solution of 4-toluenesulfonic acid monohydrate (2.50 g, 13.1 mmol, 2.5 eq.) in ACN (25 mL) was added at rt through the septum over the course of 5 min using a needle and syringe. The reaction mixture was stirred at rt for 5 h and was subsequently neutralized using a stream of gaseous ammonia. The reaction mixture was then adsorbed onto silica (20 g) and the crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (10-43%). The amine 30 (1.36 g, 44%; R.sub.f 0.46 in D2 on an NH.sub.3-pretreated TLC plate, visualization by KMnO.sub.4) was obtained as a yellow oil. HRMS (ESI): m/z calcd. for C.sub.36H.sub.64N.sub.2O.sub.4 [M+H].sup.+ 589.4939; found 589.4937.

    Hexakis((Z)-3,7-dimethylocta-2,6-dien-1-yl) cis,cis-5,5,5,5,5 ,5-((((cyclohexane-1,3,5-tricar-bonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azanetriyl))hexapentanoate 31

    [0232] Thionylchloride (700 ?L) and DMF (6 ?L) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (65 mg, 301 ?mol), and the suspension was stirred for 3 h at 70? C. in a closed vial; during this time the suspension turned into a clear solution. Excess SOCl.sub.2 was removed at 70? C. with a stream of dry nitrogen, the residue was dried at rt in vacuo (20 min), and the obtained solid was dissolved under argon in anhydrous TCE (2 mL) and DIPEA (524 ?L, 3.01 mmol, 10 eq.). Then, a solution of amine 30 (796 mg, 1.35 mmol, 4.5 eq.) in anhydrous TCE (2.0 mL) was added and the reaction mixture was stirred for 30 min at rt. The reaction mixture was then evaporated in vacuo, redisolved in DCM, adsorbed onto silica (10 g), and the DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 5-29%) to yield the target compound 31 (351 mg, 51%) as a yellow waxy semi-solid. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=5.69, 5.34, 5.08, 4.55, 3.21, 2.41, 2.38, 2.30, 2.22, 2.10, 2.06, 1.75, 1.67, 1.60, 1.59, 1.58, 1.47, 1.28 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=173.81, 173.58, 142.44, 132.12, 123.56, 119.22, 60.96, 53.82, 53.48, 44.12, 39.44, 34.17, 32.15, 31.85, 29.50, 27.07, 26.75, 26.63, 25.68, 23.50, 22.91, 17.65 ppm. HRMS (MALDI): m/z calcd. for C.sub.117H.sub.199N.sub.6O.sub.15 [M+H].sup.+ 1928.4988; found 1928.4963.

    Example 10

    Tetra(octan-2-yl) cis,cis-6,6,6,6-((((5-(methoxycarbonyl)cyclohexane-1,3-dicarbonyl) bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate 32

    [0233] Thionylchloride (700 ?L) and DMF (6 ?L) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (60 mg, 278 ?mol), and the suspension was stirred for 3 h at 70? C. in a closed vial; during this time the suspension turned into a clear solution. Excess SOCl.sub.2 was removed at 70? C. with a stream of dry nitrogen, the residue was dried at rt in vacuo (20 min), and the obtained solid was dissolved under argon in anhydrous TCE (2 mL) and DIPEA (483 ?L, 2.78 mmol, 10 eq.). Then, a solution of amine 18 (632 mg, 1.11 mmol, 4 eq.) in anhydrous TCE (1.0 mL) was added and the reaction mixture was stirred for 40 min at rt. The reaction mixture was then evaporated in vacuo, redisolved in DCM, adsorbed onto silica (10 g), and the DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 5-29%) to yield compound 32 (242 mg, 65%; R.sub.f 0.68 in D2 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as a yellow oil. The product 32 is a result of imperfect MeOH removal from amine 18. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=5.68, 5.29, 4.88, 3.67, 3.22, 2.43, 2.40, 2.27, 2.20, 2.19, 2.18, 2.09, 1.62, 1.59, 1.57, 1.56, 1.48, 1.47, 1.46, 1.30, 1.28, 1.27,1.19, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=174.64, 173.75, 173.38, 70.83, 53.82, 53.76, 51.84, 43.99, 42.09, 39.43, 35.93, 34.66, 31.81, 31.73, 31.20, 29.50, 29.09, 27.06, 26.72, 25.36, 24.99, 22.56, 20.00, 14.05 ppm.

    [0234] HRMS (MALDI): m/z calcd. for C.sub.78H.sub.146N.sub.4O.sub.12 [M+H].sup.+ 1332.1010; found 1332.0987.

    Example 11

    Tetrakis(3-methylhexyl) cis,cis-7,7,7,7-((((5-(methoxycarbonyl)cyclohexane-1,3-dicarbonyl) bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetraheptanoate 33

    [0235] Thionylchloride (700 ?L) and DMF (6 ?L) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (55 mg, 254 ?mol), and the suspension was stirred for 3 h at 70? C. in a closed vial; during this time the suspension turned into a clear solution. Excess SOCl.sub.2 was removed at 70? C. with a stream of dry nitrogen, the residue was dried at rt in vacuo (20 min), and the obtained solid was dissolved under argon in anhydrous TCE (2 mL) and DIPEA (443 ?L, 2.54 mmol, 10 eq.). Then, a solution of MeOH (10.3 ?L, 254 ?mol, 1.0 eq.) in TCE (0.5 mL) was added at 0? C. The reaction mixture was allowed to warm to room temperature and after stirring for 1 h, amine 22 (376 mg, 661 ?mol, 2.6 eq.) in anhydrous TCE (2.0 mL) was added and the reaction mixture was stirred for 40 min at rt. The reaction mixture was then evaporated in vacuo, redisolved in DCM, adsorbed onto silica (10 g), and the DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 5-29%) to yield the target compound 33 (87 mg, 26%; R.sub.f 0.75 in D2 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as a pale-yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=5.68, 5.29, 4.09, 3.67, 3.22, 2.43, 2.40, 2.28, 2.20, 2.18, 2.09, 1.64, 1.61, 1.57, 1.53, 1.48, 1.45, 1.42, 1.34, 1.31, 1.28, 1.13, 0.89, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=174.65, 173.91, 173.76, 62.85, 53.85, 51.85, 43.98, 42.09, 39.42, 39.15, 35.52, 34.22, 31.89, 31.19, 29.55, 29.51, 29.06, 27.20, 27.10, 26.72, 24.95, 19.94, 19.47, 14.26 ppm.

    [0236] HRMS (MALDI): m/z calcd. for C.sub.78H.sub.147N.sup.4O.sub.12 [M+H].sup.+ 1332.1010; found 1332.0984.

    Example 12

    Compound 34

    [0237] Thionylchloride (1.2 mL) and DMF (10 ?L) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (160 mg, 740 ?mol), and the suspension was stirred for 3 h at 70? C. in a closed vial; during this time the suspension turned into a clear solution. Excess SOCl.sub.2 was removed at 70? C. with a stream of dry nitrogen, the residue was dried at rt in vacuo (20 min), and the obtained solid was dissolved under argon in anhydrous TCE (3 mL) and DIPEA (1.29 mL, 7.40 mmol, 10 eq.). Then, a solution of BnOH (76 ?L, 740 ?mol, 1.0 eq.) in TCE (0.5 mL) was added at 0? C. The reaction mixture was allowed to warm to room temperature and after stirring for 2 h, amine 18 (1.09 g, 1.92 mmol, 2.6 eq.) in anhydrous TCE (3.0 mL) was added and the reaction mixture was stirred for 40 min at rt. The reaction mixture was then evaporated in vacuo, redisolved in DCM, adsorbed onto silica (10 g), and the DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 5-29%) to yield the compound 34 (150 mg, 14%; R.sub.f 0.67 in D2 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as a pale-yellow oil. HRMS (MALDI): m/z calcd. for C.sub.84H.sub.15,N.sub.4O.sub.12 [M+H].sup.+ 1408.1323; found 1408.1283.

    cis,cis-3,5-Bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)cyclohexane-1-carboxylic acid 35

    [0238] A pear-shaped flask was equipped with a magnetic stir bar and loaded with palladium on carbon (10% Pd/C, 25 mg). The flask was closed with a rubber septum, connected to a Schlenk line via a needle, and the atmosphere in the flask was exchanged for argon via 3 cycles of vac-Ar. Subsequently, a solution of compound 34 (150 mg, 107 ?mol) in MeOH (3 mL) was added through the septum using a needle and syringe and the stirring was turned on. The flask was connected to a continuous stream of hydrogen using a second needle and the main argon inlet into the Schlenk line was closed (excess hydrogen continuously released from the flask through the Schlenk line bubbler). The reaction mixture was vigorously stirred for 4 h at rt. Then the flask was thoroughly flushed with argon and the palladium on carbon was removed by filtering through a PTFE syringe filter. The volatiles were then removed in vacuo yielding the product 35 (110 mg, 79%, R.sub.f 0.27 in D2 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as a colorless oil. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=6.59, 4.88, 3.20, 3.15, 2.71, 2.67, 2.27, 2.24, 2.20, 2.13, 2.02, 1.64, 1.63, 1.60, 1.58, 1.57, 1.56, 1.54, 1.47, 1.45, 1.32, 1.28, 1.27, 1.26, 1.18, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=180.09, 175.19, 173.10, 70.94, 53.01, 52.06, 44.36, 44.32, 39.22, 35.91, 34.43, 32.48, 32.16, 31.72, 29.15, 29.08, 26.72, 26.62, 26.30 25.36, 24.69, 24.11, 22.55, 19.99, 14.06 ppm. HRMS (MALDI): m/z calcd. for C.sub.77H.sub.145N.sub.4O.sub.12 [M+H].sup.+ 1318.0854; found 1318.0842.

    Tetra(octan-2-yl) cis,cis-6,6,6,6-((((5-(pyrrolidine-1-carbonyl)cyclohexane-1,3-dicarbonyl) bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate 36

    [0239] A vial equipped with a magnetic stir bar was was loaded with compound 35 (60 mg, 45.5 ?mol), NH.sub.4Cl (12.2 mg, 228 ?mol, 5 eq.), and flushed with argon via piercing the septum with a needle. Subsequently, anhydrous DMF (1.5 mL) and DIPEA (48 ?L, 273 ?mol, 6 eq.) were added using a needle and syringe. The resulting suspension was stirred for 10 min at rt, then a solution of PyBroP (53 mg, 114 ?mol, 2.5 eq.) in anhydrous DMF (0.5 mL) was added and the reaction mixture was stirred at rt for 90 min. The reaction mixture was then evaporated in vacuo, redisolved in DCM, adsorbed onto silica (10 g), and the DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 5-29%) to yield the compound 36 (18 mg, 29%; R.sub.f 0.42 in D2 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as the main product in the form of a pale-yellow oil. The compound 36 is a product of pyrrolidine impurity in PyBroP resulting from PyBroP hydrolysis. .sup.1H NMR (600 MHz, CDCl.sub.3): ?=6.08, 4.88, 3.48, 3.43, 3.35, 3.25, 3.26, 3.20, 3.16, 2.72, 2.50, 2.32, 2.28, 2.15, 1.98, 1.95, 1.85, 1.69, 1.68, 1.66, 1.65, 1.60, 1.57, 1.50, 1.46, 1.34, 1.28, 1.26, 1.19, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=174.39, 173.14, 172.79, 70.97, 53.37, 47.44, 47.40, 47.22, 47.19, 46.43, 46.30, 46.27, 45.90, 44.03, 41.39, 39.09, 35.92, 34.44, 32.08, 31.73, 31.20, 29.10, 26.70, 26.14, 25.37, 24.69, 24.24, 22.57, 20.00, 14.06. HRMS (MALDI): m/z calcd. for C.sub.81H.sub.152N.sub.5O.sub.11 [M+H].sup.+ 1371.1483; found 1371.1467.

    Example 13

    Trimethyl cis,cis-1,3,5-cyclohexanetricarboxylate 37

    [0240] To a solution of cis,cis-1,3,5-cyclohexanetricarboxylic acid (9.90 g, 45.8 mmol) in anhydrous methanol (150 mL) placed in a round-bottom flask equipped with a calcium chloride drying tube and magnetic stirrer was slowly added thionyl chloride (15.29 mL, 210.8 mmol, 4.6 eq.) through a rubber septum via syringe. After the violent exothermic reaction ceased, the solution was stirred at room temperature for 12 h. The solvent was then removed in vacuo, the oily residue was poured into a 500 mL separatory flask, diluted with saturated aqueous NaHCO.sub.3 (250 mL), and the product was extracted with diethyl ether (2?100 mL). The combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered through an S2 sintered glass, and the solvents were evaporated in an RVE to give 37 as a pale-yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3): ?=3.68 (s, 9H), 2.44-2.32 (m, 3H), 2.31-2.21 (m, 3H), 1.59-1.45 (m, 3H) ppm. HRMS (ESI): m/z calculated for C.sub.12H.sub.19O.sub.6[M+H].sup.+ 259.11761; found 259.11766.

    Trimethyl cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylate 38

    [0241] Diisopropylamine was distilled from sodium hydroxide before use. A solution of diisopropylamine (2.19 mL, 15.5 mmol, 4 eq.) in anhydrous diethyl ether (10 mL) was added dropwise to the solution of 2.0 M n-butyllithium (7.74 mL, 15.5 mmol, 4 eq.) at 0? C. to generate lithium diisopropylamide in situ. A solution of 37 (1.00 g, 3.87 mmol; 1 eq.) in anhydrous diethyl ether (10 mL) was added dropwise to the reaction solution at 0? C., and the mixture was stirred for 2 h at 0? C. Dimethyl sulfate (2.20 mL, 23.2 mmol, 6 eq.) was added, and the stirring continued overnight at rt. The product was washed with water, 1 M HCl and brine, dried over sodium sulfate, filtered through an S2 sintered glass, and the solvents were evaporated in an RVE. GC-MS analysis exhibited that ratio of two isomers cis, cis/cis, trans=7:1 in the crude product. The fractional crystallization in the mixture of diethyl ether and pentane afforded cis, cis isomer 38 (225 mg, 19%) as white crystals. .sup.1H NMR (400 MHz, CDCl.sub.3): ?=3.65 (s, 9H), 2.74 (d, J=13.4 Hz, 3H), 1.21 (s, 9H), 0.97 (d, J=14.8 Hz, 3H) ppm. HRMS (EI): m/z calcd. for C.sub.15H.sub.24O.sub.6[M].sup.+300.1567; found 300.1565.

    cis,cis-1,3,5-Trimethylcyclohexane-1,3,5-tricarboxylic acid 39

    [0242] Trimethyl cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylate 38 (220 mg, 0.73 mmol, 1 eq.) was dissolved in methanol (3.0 mL). A solution of lithium hydroxide monohydrate (369 mg, 6.59 mmol, 9 eq.) in water (2.0 mL) was added, and the reaction mixture was stirred at rt overnight. Methanol was removed by evaporation in an RVE. The solution was cooled in an ice bath, and the pH was adjusted to 1 with concentrated HCl. A white precipitate formed immediately and was collected by suction filtration to yield 39 (60 mg, 32%). .sup.1H NMR (400 MHz, DMSO-d6): ?=11.96 (br s, 3H), 2.52 (d, 3H, partial overlap with solvent signal), 1.20 (d, J=14.7 Hz, 3H), 1.19 (s, 9H) ppm.

    Hexakis(3-methylhexyl) cis,cis-7,7,7,7,7 ,7-((((1,3,5-trimethylcyclohexane-1,3,5-tricar-bonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azanetriyl))hexaheptanoate 40

    [0243] Thionylchloride (0.50 mL) and DMF (2 ?L) was added to cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid 39 (34 mg, 0.13 mmol), and the suspension was stirred overnight at 70? C. in a closed vial. Excessive SOCl.sub.2 was blown out with a stream of dry nitrogen, the residue was dried in vacuo (10 min), and after cooling down to rt the residue was dissolved in anhydrous TCE (0.5 mL). Then, a solution of amine 22 (337 mg, 0.59 mmol, 4.5 eq.) and DIPEA (229 ?L, 1.32 mmol, 10 eq.) in anhydrous TCE (1.00 mL) was added, and the reaction mixture was stirred for 15 min at rt. The reaction mixture was then adsorbed onto silica (4 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (40 g, elution with a linear gradient of D1 in DCM, 0-30%) to yield the target compound 40 (6 mg, 5%; R.sub.f 0.26 in D4, visualization by ninhydrin) as a viscous pale-yellow oil. HRMS (MALDI): m/z calcd. for C.sub.114H.sub.216N.sub.6O.sub.15 [M+H].sup.+ 1910.6396; found 1910.6368.

    Example 14

    Hexa(octan-2-yl) cis,cis-6,6,6,6,6,6-((((1,3,5-trimethylcyclohexane-1,3,5-tricarbonyl) tris(azanediyl))tris(hexane-6,1-diyl))tris(azanetriyl))hexahexanoate 41 and cis,cis-3,5-Bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)-1,3,5-trimethylcyclohexane-1-carboxylic acid 42

    [0244] Thionylchloride (0.50 mL) and DMF (2 ?L) was added to 39 (28 mg, 0.11 mmol), and the suspension was stirred for 2 h at 70? C. in a closed vial. Excessive SOCl.sub.2 was blown out with a stream of dry nitrogen, the residue was dried in vacuo (10 min), and after cooling down to rt the residue was dissolved in anhydrous TCE (0.25 mL). Then, a solution of amine 18 (278 mg, 0.49 mmol, 4.5 eq.) and DIPEA (180 ?L, 1.08 mmol, 10 eq.) in anhydrous TCE (0.75 mL) was added, and the reaction mixture was stirred for 15 min at rt. The reaction mixture was then adsorbed onto silica (4 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (40 g, elution with a linear gradient of D1 in DCM, 0-30%) to yield the target compound 41 (49 mg, 24%; R.sub.f 0.30 in D4, visualization by ninhydrin) as a viscous pale-yellow oil and compound 42 (52 mg, 45%; R.sub.f 0.28 in D4, visualization by ninhydrin) as a pale-yellow oil.

    [0245] 41 .sup.1H NMR (600 MHz, CDCl.sub.3): ?=7.98, 4.88, 3.12, 2.88, 2.75, 2.68, 2.27, 1.64, 1.56, 1.46, 1.45, 1.28, 1.27, 1.26, 1.23, 1.19, 1.12, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=184.26, 177.45, 173.06, 70.96, 52.45, 51.68, 43.03, 43.00, 42.38, 38.83, 35.92, 34.43, 34.17, 33.21, 31.72, 29.08, 28.95, 22.56, 19.99, 14.05 ppm. HRMS (MALDI): m/z calcd. for C.sub.114H.sub.216N.sub.6O.sub.151 [M+H].sup.+ 1910.6396; found 1910.6364. 42 HRMS (MALDI): m/z calcd. for C.sub.80H.sub.150N.sub.4O.sub.12 [M+H].sup.+ 1360.1323; found 1360.1875.

    Example 15

    Trimethyl cis,cis-1,3,5-tris((benzyloxy)methyl)cyclohexane-1,3,5-tricarboxylate 43

    [0246] Diisopropylamine was distilled from sodium hydroxide before use. A solution of diisopropylamine (6.56 mL, 46.5 mmol, 4 eq.) in anhydrous diethyl ether (30 mL) was added dropwise to the solution of 2.0 M n-butyllithium (23.2 mL, 46.5 mmol, 4 eq.) at 0? C. to generate lithium diisopropylamide in situ. A solution of trimethyl cyclohexane-1,3,5-tricarboxylate 37 (3.00 g, 11.6 mmol; 1 equiv.) in anhydrous diethyl ether (30 mL) was added dropwise to the reaction solution at 0? C., and the mixture was stirred for 2 h at 0? C. Benzyl chloromethyl ether (9.69 mL, 69.7 mmol, 6 eq.) was added, and the stirring continued over-night at rt. The product was washed with water, 1 M HCl and brine, dried over sodium sulphate, filtered through an S2 sintered glass, and the solvent was evaporated in an RVE. The crude product was purified by flash chromatography on silica (200 g, elution with a linear gradient of ethyl acetate in cyclohexane, 0-20%). The fractional crystallization in the mixture of diethyl ether (3.0 mL) and pentane (18.0 mL) afforded cis, cis isomer 43 (1.52 mg, 22%) as white crystals. .sup.1H NMR (400 MHz, CDCl.sub.3): ?=7.36-7.22 (m, 15H, partial overlap with solvent signal), 4.46 (s, 6H), 3.69 (s, 9H), 3.39 (s, 6H), 2.68 (d, J=14.2 Hz, 3H), 1.18 (d, J=14.9 Hz, 3H) ppm. HRMS (ESI): m/z calcd. for C.sub.36H.sub.42O.sub.9[M+Na].sup.+ 641.2721; found 641.2719.

    cis,cis-1,3,5-Tris((benzyloxy)methyl)cyclohexane-1,3,5-tricarboxylic acid 44

    [0247] Compound 43 (250 mg, 0.40 mmol, 1 eq.) was dissolved in methanol (4.0 mL). A solution of lithium hydroxide monohydrate (152 mg, 3.64 mmol, 9 eq.) in water (2.0 mL) was added, and the reaction mixture was stirred at rt overnight. Methanol was removed by evaporation in an RVE. The solution was cooled in an ice bath, and the pH was adjusted to 1 with concentrated HCl. A white precipitate formed immediately and was collected by suction filtration to yield 44 (200 mg, 86%). .sup.1H NMR (400 MHz, DMSO-d6): ?=12.12 (br s, 3H), 7.37-7.18 (m, 15H), 4.39 (s, 6H), 3.39 (s, 6H), 2.36 (d, J=14.9 Hz, 3H), ppm.

    Tetra(octan-2-yl) cis,cis-6,6,6,6-((((1,3,5-tris((benzyloxy)methyl)-5-(bis(6-(octan-2-yloxy)-6-oxohexyl)carbamoyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl)) bis(azanetriyl))tetrahexanoate 45 and cis,cis-1,3,5-Tris((benzyloxy)methyl)-3,5-bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)cyclohexane-1-carboxylic acid 46

    [0248] Thionylchloride (0.50 mL) and DMF (2 ?L) was added to 44 (100 mg, 0.17 mmol, 1 eq.), and the suspension was stirred overnight at 70? C. in a closed vial. Excessive SOCl.sub.2 was blown out with a stream of dry nitrogen, the residue was dried in vacuo (10 min), and after cooling down to rt the residue was dissolved in anhydrous TCE (0.5 mL). Then, a solution of amine 18 (444 mg, 0.78 mmol, 4.5 eq.) and DIPEA (302 ?L, 1.73 mmol, 10 eq.) in anhydrous TCE (1.50 mL) was added, and the reaction mixture was stirred for 15 min at rt. The reaction mixture was then adsorbed onto silica (4 g), and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography on silica (80 g, elution with a linear gradient of D1 in DCM, 0-20%) to yield the target compound 45 (46 mg, 12%; R.sub.f 0.46 in D4, visualization by ninhydrin) as a viscous pale-yellow oil and compound 46 (37 mg, 13%; R.sub.f 0.39 in D4, visualization by ninhy-drin) as a pale-yellow oil.

    [0249] 45 HRMS (MALDI): m/z calcd. for C.sub.135H.sub.234N.sub.6O.sub.18 [M+H].sup.+ 2228.7652; found 2228.7702.

    [0250] 46 HRMS (MALDI): m/z calcd. for C.sub.101H.sub.168N.sub.4O.sub.15 [M].sup.+1677.2506; found 1677.2479.

    Hexa(octan-2-yl) cis,cis-6,6,6,6,6,6-((((1,3,5-tris(hydroxymethyl)cyclohexane-1,3,5-tricar-bonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azanetriyl))hexahexanoate 47

    [0251] Following the procedure outlined for 35, the target compound 47 was prepared from lipidoid 45 (20 mg, 0.008 mmol, 1 eq.), palladium on carbon (10% Pd/C, 19 mg, 0.018 mmol, 2 eq.) and acetic acid (80 ?L, 1.73 mmol) to yield lipidoid 47 (5 mg, 30%) as a thick pale-yellow oil. HRMS (MALDI): m/z calcd. for C.sub.114H.sub.216N.sub.6O.sub.18 [M+H].sup.+ 1958.6244; found 1958.6314.

    cis,cis-3,5-Bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)-1,3,5-tris(hydroxyme-thyl)cyclohexane-1-carboxylic acid 48

    [0252] Following the procedure outlined for 35, the target compound 48 was prepared from lipidoid 46 (15 mg, 0.009 mmol, 1 eq.), palladium on carbon (19 mg, 0.018 mmol, 2 eq.) and acetic acid (80 ?L, 1.73 mmol) to yield lipidoid 48 (7 mg, 59%) as athick pale-yellow oil. HRMS (MALDI): m/z calcd. for C.sub.80H.sub.150N.sub.4O.sub.15 [M].sup.+1407.1098; found 1407.1042.

    Example 16

    Tetra(octan-2-yl) cis,cis-6,6,6,6-((((1,3,5-tris((benzyloxy)methyl)-5-(pyrrolidine-1-carbonyl) cy-clohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate 49

    [0253] Compound 44 (100 mg, 0.17 mmol, 1 eq.) was dissolved in anhydrous DMF (1.0 mL) and DMAP (3 mg, 0.02 mmol, 0.15 eq.) and DIPEA (450 ?L, 2.60 mmol, 10 eq.) were added. A solution of PyBroP (363 mg, 0.78 mmol, 4.5 eq.) in anhydrous DMF (1.0 mL) was added dropwise, and the reaction mixture was stirred for 1.5 h at rt under argon atmosphere. Then, a solution of amine 18 (444 mg, 0.78 mmol, 4.5 eq.) in anhydrous DMF (1.0 mL) was added, and the reaction mixture was stirred for 1 h at rt under argon atmosphere. The reaction mixture was then adsorbed onto silica (4 g), and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography on silica (80 g, elution with a linear gradient of D1 in DCM, 0-30%) to yield the target compound 49 (146 mg, 51%; R.sub.f 0.58 in D3, visualization by ninhydrin) as a pale-yellow oil. HRMS (MALDI): m/z calcd. for C.sub.105H.sub.175N.sub.5O.sub.14 [M+Na].sup.+1753.3028; found 1753.3044.

    Tetra(octan-2-yl) cis,cis-6,6,6,6-((((1,3,5-tris(hydroxymethyl)-5-(pyrrolidine-1-carbonyl)cyclo-hexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate 50

    [0254] Following the procedure outlined for 35, the target compound 50 was prepared from lipidoid 49 (70 mg, 0.04 mmol, 1 eq.), palladium on carbon (86 mg, 0.40 mmol, 2 eq.) and acetic acid (80 ?L, 1.73 mmol) to yield lipidoid 50 (57 mg, 96%) as a thick pale-yellow oil. HRMS (MALDI): m/z calcd. for C.sub.84H.sub.157N.sub.5O.sub.14 [M+Na].sup.+1483.1619; found 1483.1656.

    Example 17

    cis,cis-N.sup.1,N.sup.7-Bis(6-(didodecylamino)hexyl)-5,7-bis(hydroxymethyl)-4-oxo-3-oxabicyclo [3.3.1]nonane-1,7-dicarboxamide 52

    [0255] PyBroP (0.323 g, 0.694 mmol, 5 eq.), N,N-diisopropylethylamine (DIPEA, 0.483 mL, 2.77 mmol, 20 eq.) and amine 10 (0.314 g, 0.694 mmol, 5 eq.) were added to a solution of triacid 44 (80 mg, 0.139 mmol) in anhydrous TCE (4 mL), and the reaction mixture was stirred at rt for 12 h. The reaction mixture was then adsorbed onto silica (10 g), and the solvents were evaporated in vacuo. The crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (20-80%). Diamide 51 (16 mg, 8.0%; R.sub.f0.56 in mobile phase D3, detection with ninhydrin) was obtained in the form of a viscous yellowish oil.

    [0256] Palladium on charcoal (10%) was added to a solution of diamide 51 in a methanol-ethyl acetate mixture (10+10 mL) under argon atmosphere. The reaction mixture was then stirred under hydrogen atmosphere at rt for 12 h. The suspension was filtered through a pad of celite and evaporated to yield lipidoid 52 as a colorless oil (5.5 mg, 42.9%, R.sub.f0.61 in mobile phase D3, detection with ninhydrin) HRMS (MALDI): m/z calcd. for C.sub.72H.sub.141N.sup.4O.sub.6 [M+H].sup.+ 1158.0846; found 1158.0822.

    Example 18

    6-(Didodecylamino)hexanoic acid 53

    [0257] A 500 mL round-bottom flask equipped with a calcium chloride drying tube and magnetic stirrer was filled with a solution of 6-aminohexanoic acid (2.00 g, 15.2 mmol) in ACN (150 mL), and sodium triacetoxyborohydride (12.93 g, 61.0 mmol, 4 eq.) was added. With intensive stirring, n-dodecylaldehyde (10.16 mL, 45.7 mmol, 3 eq.) was slowly added through a rubber septum via syringe, and the resulting white suspension was stirred at room temperature for 5 d. The reaction mixture was then adsorbed onto silica (25 g), and the solvents were evaporated in vacuo. The crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (0-100%). Amino acid 53 (3.203 g, 44.9%) was obtained as a colourless oil. .sup.1H NMR (400 MHz, CDCl.sub.3): ?=2.88-2.84 (m, 6H), 2.24 (t, 2H), 1.62-1.72 (m, 2H), 1.42-1.26 (m, 44H) 0.89 (t, 6H) ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): ?=178.65, 51.37, 36.22, 31.90, 29.61-29.20, 27.07, 26.74, 25.45, 23.78, 23.31, 22.67, 14.10 ppm. HRMS (ESI): m/z calculated for C.sub.30H.sub.60O.sub.2N [M?H].sup.? 466.46295; found 466.46286.

    cis,cis-1,3,5-Triaminocyclohexane trihydrobromide 55

    [0258] The synthesis was carried out according to Bowen, T. et al., Bioorganic & Medicinal Chemistry Letters, Vol. 6, No. 7, 1996, 807-810 and slightly modified. cis,cis-1,3,5-Cyclohexanetricarboxylic acid (2.0 g, 9.25 mmol) was suspended in toluene (75 mL), and DIPEA (4.83 mL, 27.75 mmol, 3 eq.) was added followed by diphenyl phosphoryl azide (5.97 mL, 27.75 mmol, 3 eq.). The mixture was stirred for 0.5 h at rt and then heated to 90? C. for 0.5 h. Benzyl alcohol (3.20 mL, 30.81 mmol, 3.33 eq.) was added and the solution was heated to 90? C. for 18 h. After cooling to rt, the product was collected by vacuum filtration, washed with minimal cold toluene, and dried under vacuum to yield 0.386 g (7.85%) of cis,cis-1,3,5-tri(N-benzyloxycarbonyl)cyclohexane 54.

    [0259] Compound 54 (0.386 g, 0.726 mmol) was suspended in a 33% solution of HBr in acetic acid (5 mL), and stirred for 12 h at rt. The yellow suspension was then diluted with diethyl ether (20 mL), and filtered through an S2 sintered glass to give trihydrobromide 55 as a yellow solid (0.248 g, 91.8%).

    cis,cis-N,N,N-(Cyclohexane-1,3,5-triyl)tris(6-(didodecylamino)hexanamide) 56

    [0260] PyBroP (0.752 g, 1.61 mmol, 6 eq.), DIPEA (0.937 mL, 5.38 mmol, 20 eq.) and acid 53 (0.755 g, 1.61 mmol, 6 eq.) were added to a solution of triamine salt 55 (0.100 g, 0.269 mmol) in a mixture of anhydrous TCE (3 mL) and anhydrous DMF (3 mL), and the reaction mixture was stirred at rt for 12 h. The reaction mixture was then adsorbed onto silica (10 g), and the solvents were evaporated in vacuo. The crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (20-30%). Lipidoid 56 (113 mg, 28.4%; R.sub.f0.65 in mobile phase D2, detection with ninhydrin) was obtained in the form of a viscous yellowish oil. HRMS (ESI): m/z calculated for C.sub.96H.sub.193O.sub.6N.sub.3 [M+H].sup.+ 1478.5134; found 1478.5162.

    Example 19

    Preparation of Transfection Reagents

    [0261] Reagents were generated by mixing the individual components listed in Table 1 up to Table 4. All tables contain the final molar concentrations in the transfection reagent. Stock 5 mM solutions of the individual components in 99.7% ethanol (v/v) were used for the preparation of A01 to A16, A21, A22, A24, A28, and A29. Stock 5 mM solutions of lipidoids, DOPE and DMG-PEG2000, and stock 10 mM solution of cholesterol, all in 99.7% ethanol (v/v) were used for the preparation of A17 to A20, A23, and A25 to A27. The DOPE-Cy5 stock solution had a concentration of 0.79 mM and was prepared in chloroform. TT3 is a lipid according to WO 2016/187531, used herein for comparison. Benchmark D-Lin-MC3-DMA lipid (Med-ChemExpress Europe) was also used for comparison.

    TABLE-US-00001 TABLE 1 Composition of transfection reagents A01-A06. Concentration of individual components in Transfection reagents (mM) Compound A01 A02 A03 A04 A05 A06 3 1.1 7 1.1 8 1.1 11 1.1 15 1.1 TT3 1.1 cholesterol 2.18 2.18 2.18 2.18 2.18 2.18 DOPE 1.64 1.64 1.64 1.64 1.64 1.64 DMG- 0.075 0.075 0.075 0.075 0.075 0.075 PEG.sub.2000

    TABLE-US-00002 TABLE 2 Composition of transfection reagents A07-A12. Concentration of individual components in transfection reagents (mM) Compound A07 A08 A09 A10 A11 A12 3 1.1 7 1.1 8 1.1 11 1.1 15 1.1 TT3 1.1 cholesterol 2.18 2.18 2.18 2.18 2.18 2.18 DOPE 1.64 1.64 1.64 1.64 1.64 1.64 DMG- 0.075 0.075 0.075 0.075 0.075 0.075 PEG.sub.2000 DOPE-Cy5 1.19 ? 10.sup.?3 1.19 ? 10.sup.?3 1.19 ? 10.sup.?3 1.19 ? 10.sup.?3 1.19 ? 10.sup.?3 1.19 ? 10.sup.?3

    TABLE-US-00003 TABLE 3 Composition of transfection reagents A13-A22. Concentration of individual components in transfection reagents (mM) Compound A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 19 1.1 23 1.1 27 1.1 31 1.1 32 1.64 33 1.64 35 1.64 36 1.64 40 1.1 41 1.1 cholesterol 2.18 2.18 2.18 2.18 2.18 2.18 2.18 2.18 2.18 2.18 DOPE 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 DMG- 0.075 0.075 0.075 0.075 0.075 0.075 0.075 0.075 0.075 0.075 PEG.sub.2000

    TABLE-US-00004 TABLE 4 Composition of transfection reagents A23-A29. Concentration of individual components in transfection reagents (mM) Compound A23 A24 A25 A26 A27 A28 A29 42 1.64 47 1.1 48 1.64 50 1.64 52 1.64 56 1.1 D-Lin-MC3-DMA 2.5 cholesterol 2.18 2.18 2.18 2.18 2.18 2.18 1.93 DOPE 1.64 1.64 1.64 1.64 1.64 1.64 DSPC 0.5 DMG-PEG.sub.2000 0.075 0.075 0.075 0.075 0.075 0.075 0.075

    Example 20

    Preparation of Lipid Nanoparticles (LNP) Containing Nucleic Acids

    [0262] The siRNA-containing LNPs (siRNA-LNPs) were prepared as follows: 300 ?l of a solution of each of the A01-A06 transfection reagents prepared in Example 19 was mixed with a solution of 1.2 nmol siRNA (catalog number 4392420, Ambion) in 300 1d 10 mM citrate buffer (pH 3.0) using a Y microfluidic device with two inputs and one output for sampling. The lipid mixture and the siRNA solution were injected sep-arately into each inlet by a linear pump at a constant flow rate of 300 l/min. The resulting 600 1d nanoparticle solution was collected and immediately diluted by the addition of 600 ?l PBS; the corresponding nanoparticle samples designated B01-B06 were thus formed from the transfection reagents A01-A06. DNA encoding the fluorescent protein mKate2 was amplified from the plasmid pmKate2-C(Evrogen) using the primers (5-ATCAACATATGGTGAGCGAGCTG-3 (SEQ ID NO. 1); 5-AAGAATTCCTATCATCTGTGCCCCAG-3 (SEQ ID NO. 2)) and cloned into the pET24a vector (Invitrogen) under the T7 promoter. Messenger RNA (mRNA) encoding mKate2 was transcribed in vitro using the Ampliscribe T7-Flash transcription kit (Lucigen) according to the manufacturer's protocol. The RNA cap analog ARCA (Jena Bioscience) was added to the in vitro transcription reaction, and the poly(A) terminus was synthesized using poly(A) polymerase (New England Biolabs) according to the standard protocol.

    [0263] The mRNA-containing LNPs (mRNA-LNPs) were prepared as follows: 300 ?l of a solution of each of the A07-A29 transfection reagents prepared in Example 19 were mixed with a solution of 120 ?g of mRNA in 300 1d of 10 mM citrate buffer (pH 3.0), the preparation was then analogous to siRNA-LNP. Thus, the corresponding nanoparticle samples designated B07-B29 were generated from the transfection reagents A07-A29. Nanoparticle samples designated B08a and Bl1a were generated from the transfection reagents A02 and A05, respectively.

    [0264] Each of the LNP samples (B01-B29) was prepared in triplicate. The hydrodynamic diameter of freshly formed LNPs was measured using dynamic light scattering (NanoZS Zetasizer, Malvern, Worcestershire, UK) at a scattering angle of 1730 at 25? C. The hydrodynamic diameter of siRNA-LNPs ranged from 67 to 110 nm, and the hydrodynamic diameter of mRNA-LNPs ranged from 82 to 288 nm (Tab. 5). In this form, the particles were used for subsequent biological tests.

    TABLE-US-00005 TABLE 5 Hydrodynamic diameter of LNPs including standard deviation measured by dynamic light scattering. LNP Diameter (nm) B01 83.3 ? 4.0 B02 109.9 ? 12.7 B03 100.1 ? 6.3 B04 81.7 ? 8.8 B05 72.3 ? 13.2 B06 67.1 ? 3.5 B07 90.4 ? 1.3 B08 153.5 ? 11.9 B09 117.0 ? 7.6 B10 90.5 ? 3.9 B11 157.7 ? 3.5 B12 81.5 ? 5.4 B13 174.3 ? 7.8 B14 144.6 ? 17.8 B15 124.5 ? 14.3 B16 143.2 ? 6.2 B17 190.8 ? 2.5 B18 190.4 ? 2.1 B19 194.8 ? 5.2 B20 194.4 ? 6.2 B21 179.4 ? 10.5 B22 261.0 ? 11.4 B23 221.0 ? 12.7 B24 217.1 ? 13.8 B25 287.8 ? 10.5 B26 165.1 ? 11.9 B27 242.1 ? 3.9 B28 227.9 ? 10.5 B29 119.1 ? 4.5

    Example 21

    Efficiency of siRNA and mRNA Incorporation into Lipid Nanoparticles

    [0265] The efficiency of packaging ofthe siRNA prepared in Example 20 (which caused the degradation of mRNA encoding tyrosyl-DNA phosphodiesterase (TDP2)) into siRNA-LNP B01-B06 was determined using a Qubit microRNA Assay Kit (Life Technologies) according to the manufacturer's protocol. The efficiency of the packaging of the mRNA (encoding the fluorescence protein mKate2) into mRNA-LNP B07-B29 prepared in Example 20 was determined using a Qubit RNA HS Assay Kit (Life Technologies) according to the manufacturer's protocol. The efficiency of incorporation was determined by comparing the concentration of siRNA and mRNA freely available in the nanoparticle solution and the concentration of siRNA and mRNA released from the nanoparticles after their decomposition. LNPs were decomposed with buffer containing Triton X-100 (10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA, 2% Triton X-100). High packaging efficiency of siRNA was demonstrated, ranging from 76 to 91%. The packaging efficiency of mRNA ranged from 60 to 96% (Tab. 6).

    TABLE-US-00006 TABLE 6 Efficiency of packaging siRNA against tyrosyl-DNA phosphodiesterase 2 (TDP2) and efficiency of packaging mRNA encoding fluorescent protein mKate2 into LNPs, including standard deviations from triplicates. LNP siRNA packaging (%) B01 76.1 ? 4.9 B02 91.1 ? 1.3 B03 77.4 ? 4.6 B04 85.0 ? 6.9 B05 75.6 ? 2.21 B06 89.9 ? 0.1 LNP mRNA packaging (%) B07 88.3 ? 1.6 B08 86.2 ? 0.9 B09 70.4 ? 6.8 B10 86.9 ? 0.6 B11 64.3 ? 3.4 B12 83.2 ? 3.2 B13 85.5 ? 0.5 B14 94.0 ? 0.1 B15 96.0 ? 1.0 B16 86.5 ? 6.5 B17 75.0 ? 11.0 B18 65.1 ? 0.1 B19 67.0 ? 2.8 B20 64.2 ? 0.2 B21 65.0 ? 0.1 B22 60.8 ? 3.2 B23 63.7 ? 0.4 B24 68.7 ? 3.3 B25 78.8 ? 1.3 B26 61.1 ? 1.1 B27 75.0 ? 0.1 B28 70.0 ? 0.1 B29 77.0 ? 2.0

    Example 22

    Cellular Toxicity of LNPs

    [0266] A human cell line derived from embryonic kidney cells expressing SV40 large T antigen (HEK293T), and human hepatocyte carcinoma cell line (HepG2) were cultured in 96-well plates (5?10.sup.4 cells in 100 1d of culture medium per well) in Dulbecco's modified medium (DMEM) supplemented with 10% foetal bovine serum (FBS) at 37? C. in 5% CO.sub.2. Cells were transfected with 2 ?l of LNPs B07-B12 generated in triplicates in Example 20 (the final total concentration of all lipid components in the well was 20 ?M) or 10 ?l of LNPs (the final total concentration of all lipid components in the well was 100 ?M) and subsequently incubated for 24 hours. The cytotoxicity of LNPs was analyzed in a CellTiterGlo 2.0 cell viability assay (Promega, USA). Cell viability was normalized to non-transfected cells (control). The results are summarized in Tables 7 and 8.

    [0267] For both cell lines used, none of the new LNPs exhibited significant toxicity compared to the 100 ?M mixture with TT3 lipid, with which the viability of HEK293T and HepG2 cells is reduced to only 27 and 15% (Tab. 8).

    TABLE-US-00007 TABLE 7 Cytotoxicity of LNPs expressed as cell viability (%) after the addition of 20 ?M transfection mixture B07-B12 for individual cell line types. LNP HEK293T HepG2 Control 100.00 ? 4.29 100.00 ? 3.44 B07 91.85 ? 4.34 93.49 ? 4.25 B08 95.90 ? 6.14 100.85 ? 6.35 B09 102.19 ? 5.75 103.10 ? 7.13 B10 100.49 ? 3.12 100.93 ? 5.87 B11 103.51 ? 5.79 101.74 ? 6.09 B12 102.55 ? 5.67 104.55 ? 4.98

    TABLE-US-00008 TABLE 8 Cytotoxicity of LNPs expressed as cell viability (%) after the addition of 100 ?M transfection mixture B07-B12 for individual cell line types. LNP HEK293T HepG2 Control 100.00 ? 4.29 100.00 ? 3.44 B07 99.86 ? 3.95 107.75 ? 7.27 B08 103.07 ? 2.05 117.75 ? 3.71 B09 106.79 ? 2.32 113.40 ? 5.38 B10 70.88 ? 3.77 95.77 ? 3.37 B11 108.84 ? 3.31 110.43 ? 1.19 B12 27.29 ? 2.38 14.92 ? 4.19

    Example 23

    Transfection of siRNA Using New LNPs In Vitro

    [0268] The siRNA-LNP particles containing small interfering RNA (siRNA, catalog number 4392420, Ambion) that causes degradation of mRNA encoding tyrosyl-DNA phosphodiesterase 2 (TDP2) were prepared according to Example 20. Lipofectamine RNAiMax (Invitrogen) was used as a control transfection reagent specifically for siRNA transfection. The human cell line HEK293T and the cell line derived from human multiple myeloma, which is very difficult to transfect with available transfection reagents, were used for siRNA-LNP knock-down (Brito J. L. R., Brown N., Morgan G. J. (2010) The transfection of siRNAs in Multiple Myeloma Cell Lines. In. Min WP., Ichim T. (eds) RNA Interference. Methods in Molecular Biology (Methods and Protocols), vol 623. Humana Press). Cells were cultured in 96-well plates (5?10.sup.4 cells in 100 1d culture medium per well) in DMEM medium supplemented with 10% FBS at 37? C. in 5% CO.sub.2. Cells were transfected with 2 1d siRNA-LNP (with a final total concentration of all lipid components of 20 ?M and a final siRNA concentration of 16 nM) and subsequently incubated for 24 hours. Transfections were performed in biological triplicates. RNA was isolated using an RNAeasy Plus Micro Kit (Qiagen). cDNA was prepared using TATAA GrandScript cDNA Supermix (TATAAbiocenter) according to the manufacturer's recommendations. Quantitative RT-PCR was performed using a LightCycler 480 (Roche Life Science). The primers for amplifying mRNA encoding TDP2 were as follows: 5-CGAGAGGAGGGTCTCAAAGAG-3 (SEQ ID NO. 3) and 5-ATTTCGGGAAGGCTGCTGTC-3 (SEQ ID NO. 4). mRNA encoding GAPDH was used to normalize the data (Primers: 5-AATCCCATCACCATCTTCCA-3 (SEQ ID NO. 5) and 5-TGGACTCCACGACGTACTCA-3 (SEQ ID NO. 6)).

    [0269] In all these cases, the new siRNA-LNPs significantly reduced the level of TDP2 mRNA in the cells compared to the commercial transfection reagent RNAiMax. In the HEK293T cell line, the new LNPs B02-B05 have almost 3-fold higher efficiency than RNAiMax. In the OPM-2 myeloma line, B03 reduces mRNA expression 13-fold better than a commercial siRNA transfection reagent (Tab. 9).

    TABLE-US-00009 TABLE 9 Reduction of endogenously expressed TDP2 mRNA in cells by new siRNA-LNPs (B01-B06) compared to commercial transfection reagent RNAiMax in the HEK293T and OPM-2 cell lines. LNP HEK293T p OPM-2 p Control 1.00 ? 0.10 1.00 ? 0.10 B01 0.24 ? 0.17 0.52 ? 0.05 B02 0.04 ? 0.02 c 0.11 ? 0.01 a B03 0.05 ? 0.03 c 0.04 ? 0.01 a B04 0.06 ? 0.02 c 0.08 ? 0.02 a B05 0.05 ? 0.01 c 0.05 ? 0.01 a B06 0.05 ? 0.01 c 0.07 ? 0.02 a RNAiMax 0.15 ? 0.06 0.52 ? 0.02 Statistics were evaluated by Student's unpaired t-test. P values are based on the Lipofectamine RNAiMax control transfection mixture; p values <0.001 are marked with the letter a, p values <0.05 are marked with the letter c.

    Example 24

    Transfection of mRNA Using New LNPs In Vitro

    [0270] A human cell line derived from embryonic kidney cells expressing the large SV40 T antigen (HEK293T), liver carcinoma cells (HepG2) and a human osteosarcoma-derived cell line (U2OS) were cultured in 96-well plates (5?10.sup.4 cells in 100 1d culture medium per well) in Dulbecco's modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37? C. in 5% CO.sub.2 Cells were transfected with 2 1d of the mRNA-LNP B07 to B12, prepared in Example 20 and subsequently incubated for 24 hours. The final total concentration of the lipid components in the well was 20 ?M and the amount of mRNA that encodes the fluorescent protein mKate2 was 100 ng. Lipofectamine? 2000 was used as a control transfection reagent. Transfections were performed in three biological replicates, with each biological replicate having three technical replicates. The Cy5 and mKate2 fluorescence was detected using the BD LSR Fortessa cytometer, and the percentage of cells positive for Cy5 fluorescence, indicating LNP entry into cells (Tab. 10), percentage of cells expressing the fluorescent protein mKate2 and the fluorescence intensity of the mKate2 (Tab. 11) were calculated. For fluorescence intensity, the data were normalized to the commercial transfection reagent Lipofectamine? 2000. According to Cy5 fluorescence, it is evident that all LNPs enter cells with an efficiency exceeding 90% (Tab. 10). Furthermore, the new LNPs B11 produce 2.13-fold more fluorescent mKate2 protein from transfected mRNA than cells transfected with Lipofectamine? 2000 transfection control (Tab. 11).

    TABLE-US-00010 TABLE 10 Transfection efficiency of new mRNA-LNPs (B07-B12) expressed as a percentage of cells positive for Cy5 for cell lines HEK293T and HepG2. The transfection mixtures contain a lipid labeled with the fluorescent dye Cy5, which makes it possible to observe the entry of LNPs into the cells. LNP HEK293T HepG2 B07 99.28 ? 0.10 98.59 ? 0.15 B08 99.19 ? 0.09 94.73 ? 0.88 B09 97.83 ? 0.63 90.52 ? 2.39 B10 97.79 ? 0.36 92.93 ? 0.77 B11 98.59 ? 0.47 96.49 ? 1.70 B12 97.94 ? 0.94 95.19 ? 0.98

    TABLE-US-00011 TABLE 11 Transfection efficiency of new mRNA-LNPs expressed as a percentage of cells expressing fluorescent mKate2 protein and fluorescence inensity of mKate2 from mRNA transfected with particles for U2OS cell line. Statistics were evaluated by Student's unpaired t-test. The p values are related to the control transfection mixture Lipofectamine2000; p values < 0.001 are indicated with the letter a. % of cells Fluorescence LNP Expressing mKate2 p of mKate2 p B07 28.76 ? 1.51 0.10 ? 0.00 B08 88.62 ? 0.60 a 1.08 ? 0.07 B09 88.38 ? 1.00 a 1.79 ? 0.23 a B10 84.46 ? 1.06 a 0.69 ? 0.08 B11 92.32 ? 2.18 a 2.13 ? 0.19 a B12 90.63 ? 0.92 a 1.86 ? 0.31 a Lip2000 47.67 ? 1.25 1.00 ? 0.05

    Example 25

    Transfection of mRNA Using New LNPs In Vitro

    [0271] A human cell line derived from embryonic kidney cells expressing the large SV40T antigen (HEK293T) was cultured in 96-well plates (2.5?10.sup.4 cells in 100 ?l culture medium per well) in Dulbecco's modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37? C. in 5% CO.sub.2. Cells were transfected with mRNA-LNPs B08a, Bl1a, B13 to B18, B20 to B25 and B29 prepared in Example 20 and then incubated for 24 hours. The final total concentration of all lipid components in the well was 20 ?M and the amount of mRNA encoding the fluorescent protein mKate2 was 100 ng. B29 LNPs containing benchmark D-Lin-MC3-DMA lipid were used as a control. Transfections were performed in triplicate. The percentage of cells expressing the fluorescent protein mKate2 and the fluorescence intensity of the mKate2 (Tab. 12) were analyzed in a BD LSR Fortessa cytometer at a a wavelength of 561-610/20 nm. For fluorescence intensity, data were normalized to the benchmark D-Lin-MC3-DMA containing LNPs (B29). It is evident that all new LNPs transfected the HEK293T cells more efficiently, as documented by significantly higher % of transfected cells as well as by significantly increased fluorescence intensity compared to the benchmark D-Lin-MC3-DMA LNPs. In particular, the new LNPs B22 produce 13.66-fold more fluorescent mKate2 protein from transfected mRNA than cells transfected with B29 LNPs containing the benchmark D-Lin-MC3-DMA lipid (Tab. 12).

    TABLE-US-00012 TABLE 12 Transfection efficiency of new mRNA-LNPs for HEK293T cell line shown as a percentage of cells expressing fluorescent mKate2 protein from mRNA delivered by LNPs. Normalized % of cells p (D-Lin- fluorescence p (D-Lin- LNP transfected MC3-DMA) of mKate2 MC3-DMA) B08a 86.2 ? 2.1 a 3.72 ? 0.11 a B11a 76.0 ? 3.8 b 3.10 ? 0.16 b B13 94.0 ? 0.7 a 5.27 ? 0.25 a B14 77.1 ? 5.3 b 1.70 ? 0.12 b B15 83.5 ? 3.8 b 1.98 ? 0.31 c B16 69.5 ? 1.3 b 1.37 ? 0.05 b B17 85.4 ? 3.2 b 3.79 ? 0.67 c B18 82.8 ? 1.5 a 3.99 ? 0.39 b B20 86.6 ? 9.1 c 7.78 ? 1.63 c B21 56.5 ? 2.3 c 2.41 ? 0.21 b B22 93.3 ? 1.5 a 13.66 ? 1.46 a B23 89.7 ? 5.7 b 7.89 ? 1.35 b B24 72.4 ? 1.3 b 1.88 ? 0.09 a B25 62.2 ? 4.2 c 1.58 ? 0.13 c B29 29.9 ? 4.2 1.00 ? 0.02 Statistics were evaluated by Student's unpaired t-test. The p values are related to the control D-Lin-MC3-DMA (B29) LNPs; p values <0.0001 are indicated with the letter a, p <0.001 as b, p <0.01 as c, respectively.

    Example 26

    Transfection Efficacy of New mRNA-LNPs Monitored by Cre-Based Recombination

    [0272] Mouse embryonic fibroblast cells (MEFs), derived from the mouse model with a global dual Cre reporter (Mazumdar, M. D.: Genesis 2007, 45:593-605), were seeded (2.5?10.sup.4 cells in 100 1d culture medium per well) on a 96-well plate and cultured in DMEM supplemented with 10% FBS at 37? C. in 5% CO, for 24 hours. Messenger RNA (mRNA) encoding Cre recombinase was prepared in vitro analogously to Example 20 from a linearized plasmid and packaged into LNP as follows: A 300 1d sample of transfection reagents A13 to A25, A02 and A27 and the control transfection reagent A29 including the benchmark D-Lin-MC3-DMA lipid, and 120 ?g of mRNA in 300 ?l of 10 mM citrate buffer (pH 3.0) were assembled into LNP using a microfluidic device analogously to Example 20. The resulting mRNA-LNPs were immediately diluted in 600 ?l PBS; the corresponding nanoparticles labeled B31 to B43 (formed from transfection reagent A13 to A25), nanoparticles B30 (formed from transfection reagent A02), nanoparticles B44 (formed from transfection reagent A27) and analogously, the benchmark nanoparticles labeled B45 were thus formed from transfection reagent A29.

    [0273] Cells were transfected with the respective mRNA-LNPs (the final total concentration of all lipid components in the well was 20 ?M) carrying 100 ng of mRNA encoding the Cre recombinase and subsequently incubated for 48 hours. B45 LNPs containing benchmark D-Lin-MC3-DMA lipid were used as a control. Transfections were performed in quadruplicates. The percentage of cells expressing GFP was analyzed using a BD LSR Fortessa cytometer (at a wavelength of 488-530/30 nm). It is again evident that all new LNPs transfected the mT/mG cells more efficiently, as documented by a significantly higher percentage of cells expressing GFP compared to cells transfected with the benchmark D-Lin-MC3-DMA LNPs (B45). Interestingly, the percentage of cells expressing GFP was always higher than 95% for all new LNPs tested (except B44), whereas LNPs containing the benchmark D-Lin-MC3-DMA lipid showed only 21% of recombined cells (Tab. 13).

    TABLE-US-00013 TABLE 13 Comparison of the efficacy of new Cre mRNA-LNPs to convert td Tomato into GFP in MEFs derived from the mT/mG reporter mouse embryos. p (D-Lin- LNP % cells expressing GFP MC3-DMA) B30 99.05 ? 0.10 a B31 99.83 ? 0.07 a B32 99.60 ? 0.01 a B33 95.50 ? 0.04 a B34 99.75 ? 0.13 a B35 99.13 ? 0.32 a B36 99.00 ? 0.17 a B37 98.85 ? 0.45 a B38 99.48 ? 0.10 a B39 95.50 ? 1.25 a B40 99.13 ? 0.22 a B41 98.00 ? 0.43 a B42 99.80 ? 0.06 a B43 99.73 ? 0.04 a B44 69.05 ? 2.00 a B45 21.25 ? 2.35 untreated 0.325 ? 0.248 a Statistics were evaluated using Student's unpaired t-test. The p values are related to the control D-Lin-MC3-DMA (B45) LNPs; p values <0.0001 are indicated with the letter a.

    Example 27

    Biodistribution of New mRNA-LNPs In Vivo

    [0274] The Messenger RNA (mRNA) encoding Cre recombinase was prepared in vitro analogously to Example 20 from a linearized plasmid and packaged into LNP as follows: A 300 ?l sample of transfection reagent A22 and the control transfection reagent A29 that includes the benchmark lipid D-Lin-MC3-DMA and 120 g of mRNA in 300 1d of 10 mM citrate buffer (pH 3.0) were assembled into LNP using a microfluidic device analogously to Example 20. The resulting mRNA-LNPs were immediately diluted in 600 ?l PBS; the corresponding B40 labeled nanoparticles were formed from the transfection reagent A22. Analogously, the benchmark nanoparticles labeled B45 were thus formed from the transfection reagent A29. The mRNA-LNPs were dialyzed in PBS and filtered. Endotoxin levels were <2 EU/ml. The mRNA-LNPs (B40 and B45) were administered intravenously at a concentration of 2.0 mg mRNA/kg in each case to 4 mice with a global dual Cre reporter (Mazumdar, M. D.: Genesis 2007, 45:593-605) (breeding BIOCEV, Vestec) enabling the analysis of successful recombination. In cells to which mRNA-LNPs carrying Cre recombinase mRNA were successfully delivered, chromosomal recombination and subsequent excision of the membrane red protein gene (the so-called red tomato) and turning on the transcription of membrane green protein (GFP) gene occurred. Mice, including non-particulate control mice, were sacrificed 5 days after particulate application, and specific organs (liver, heart, kidney, lungs, and spleen) were subjected to histological analysis according to a standardized protocol. Analysis of histological images revealed a complete distribution of the B40 nanoparticles in the liver, leading to a 50-80% conversion of cells expressing the red membrane protein to cells expressing the green membrane protein 5 days after application. In the case of the D-Lin-MC3-DMA lipid containing LNPs B45, the conversion was about 20% (FIG. 10).

    [0275] Histological analysis was further complemented by the evaluation of tdTomato/GFP conversion in the liver of the mT/mG mice by genomic DNA (gDNA) PCR analysis. Briefly, 50 ng of the gDNA template isolated from the respective liver samples was used in the PCR reaction with subsequent primers: (5-AACGTGCTGGTTATTGTGCTG-3 (SEQ ID NO. 7); 5-AAGTCGTGCTGCTTCATGTG-3 (SEQ ID NO. 8)). In FIG. 11, electrophoretic analysis of the resulting gDNA PCR amplification of the liver samples shows the PCR product of 530 bp in the case of the active Cre recombination at the tdTomato/tGFP locus, while the PCR product of 2943 bp (upper band) in the case no Cre recombination occurred. The designated B40 mRNA-LNPs showed complete Cre recombination in all 4 mice tested, while in the case of the benchark LNPs (B45) the recombination was complete only in 50% of the tested mice.

    Example 28

    Efficacy of New mRNA-LNPs In Vivo

    [0276] Messenger RNA (mRNA) encoding hEPO (human erythropoetin) was prepared in vitro analogously to Example 20 from a linearized plasmid and packaged in LNP as follows: A 300 1d sample of transfection reagent A23 or the control transfection reagent A29 that includes the benchmark lipid D-Lin-MC3-DMA and 120 ?g of mRNA in 300 ?l of 10 mM citrate buffer (pH 3.0) were assembled into LNP using a microfluidic device analogously to Example 20. The resulting mRNA-LNPs were immediately diluted in 600 ?l PBS; the corresponding B46 labeled nanoparticles were thus formed from the transfection reagent A23. Analogously, the benchmark nanoparticles labeled B47 were thus formed from the transfection reagent A29. The mRNA-LNPs were dialyzed in PBS and filtered. Endotoxin levels were <0.3 EU/ml. The mRNA-LNPs (B46 and B47) were administered intravenously at a concentration of 0.5 mg mRNA/kg in each case to 3 C57Bl/6 mice (BIOCEV, Vestec), wherein three control C57Bl/6 mice were administered with PBS. Six hours after administration, blood was collected from mice via the tail vein and allowed to clot at room temperature in serum separator tubes. The tubes were then centrifuged at 7000 rpm for 7 min and the sera samples were aliquoted and stored at ?80? C. until analysis. hEPO concentrations were determined using an hEPO ELISA assay (Catalog #DEP00; R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Hematology profiles were determined using a standardized protocol. Nanoparticles designated as B46 showed higher, yet statistically not significant, production of the human EPO protein 6 hours after administration compared to control B47 LNPs. Importantly, LNPs containing new lipidoid (B46) did not alter hematology profile as opposed to control B47 LNPs. The blood test after the new LNP administration remained comparable to PBS control (Tab. 14).

    TABLE-US-00014 TABLE 14 Comparison of the in vivo efficacy of new human EPO mRNA- LNPs in protein production six hours after administration. hEPO WBS# Neu# Lym# LNP [mIU/ml] [*10.sup.9/L] [*10.sup.9/L] [*10.sup.9/L] B46 750 ? 514 4.91 ? 0.57 0.90 ? 0.19 3.58 ? 0.73 B47 465 ? 154 10.06 ? 2.59 4.89 ? 2.66 4.23 ? 0.43 PBS 4.40 ? 1.12 1.02 ? 0.84 3.09 ? 0.33

    Example 29

    Efficacy of New siRNA-LNPs In Vivo

    [0277] Transfection reagents A02, A14, A16, A17, A22, A23 and A29 from Example 19 was used to form siRNA-LNPs designated as B48 to B54, respectively, with siRNA targeting mouse apolipoprotein B (ApoB) gene, a hepatocyte-expressed gene involved in cholesterol transport (catalogue number 238055 Apob mouse siPOOL-40 kit, siTOOLs Biotech GmbH) and alternatively siRNA-LNPs (B55 to B60) with control non-targeted siRNA-LNPs (enclosed in 238055 Apob mouse siPOOL-40 kit, siTOOLs Biotech GmbH), assembled as described in Example 20. The hydrodynamic diameter measured by dynamic light scattering ranged from 103 nm to 154 nm and the efficiency of packaging of the siRNA pool ranged from 59% to 99%. The siRNA-LNPs were dialyzed to PBS. Endotoxin levels were <2 EU/ml. Mice were fasted for 4 hours before plasma collection by retroorbital bleed. The siRNA-LNPs targeting ApoB were administered intravenously to 3 C57Bl/6 mice (BIOCEV, Czech Center of Phenogenomics, Vestec) at a concentration of 16 ?g of siRNA, wherein the control 3 mice were administered with 16 ?g of non-targeting siRNA-LNPs and another 3 mice were administered PBS control. All mice were sacrificed 2 days after LNP application. Plasma levels of cholesterol, triglycerides and LDL-C were measured by using automated systems at the Czech Center of Phenogenomics according to standardized protocol.

    [0278] Clinical biochemistry of plasma markers such as total cholesterol, triglycerides and LDL-C, affected by ApoB knock down, were significantly decreased compared to control animals, demonstrating thus the efficient delivery of ApoB siRNA by novel LNPs into the liver (Tab. 15).

    TABLE-US-00015 TABLE 15 Clinical biochemistry of plasma markers indicating efficient ApoB knockdown in the liver. Statistics were evaluated by Student's unpaired t-test. The p1 values are always relative to the control mice injected with PBS; the p2 values are always relative to the mice injected with respective (B55 to B60) LNPs with control non- targeted siRNA; values of p < 0.0001 are marked as a, p < 0.001 are marked as b, p < 0.01 are marked as c, and p < 0.05 are marked as d. Those cases that were not evaluated are marked with n.e.. Total cholesterol, Triglycerides, LDL-C, LNP mmol/L p1 p2 mmol/L p1 p2 mmol/L p1 p2 PBS control 2.27 ? 0.20 0.60 ? 0.12 0.45 ? 0.05 B48 0.82 ? 0.02 a a 0.71 ? 0.07 0.21 ? 0.02 b B49 0.52 ? 0.06 a a 0.19 ? 0.01 b b 0.15 ? 0.03 b d B50 0.85 ? 0.45 c d 0.42 ? 0.13 c 0.33 ? 0.20 d B51 0.51 ? 0.10 a b 0.25 ? 0.01 c a 0.22 ? 0.05 c d B52 0.50 ? 0.21 a b 0.27 ? 0.01 c a 0.19 ? 0.08 c d B53 0.62 ? 0.15 a b 0.3 ? 0.01 c a 0.29 ? 0.02 c B55 2.36 ? 0.61 n.e. 0.52 ? 0.01 n.e. 0.64 ? 0.14 n.e. B56 2.47 ? 0.20 n.e. 0.65 ? 0.10 n.e. 0.63 ? 0.10 d n.e. B57 2.41 ? 0.50 n.e. 0.80 ? 0.20 n.e. 0.8 ? 0.22 d n.e. B58 2.46 ? 0.76 n.e. 0.92 ? 0.14 d n.e. 0.78 ? 0.24 d n.e. B59 2.50 ? 0.77 n.e. 0.79 ? 0.08 n.e. 0.77 ? 0.32 n.e. B60 2.56 ? 0.76 n.e. 1.00 + 0.07 c n.e. 0.73 ? 0.19 d n.e.

    [0279] The toxicity of new siRNA-LNPs was further evaluated by analyzing the clinical biochemistry of plasma markers that indicate organ failure and the hematology profile compared to saline-injected controls (PBS) and control LNPs (B54) containing the lipid D-Lin-MC3-DMA. In particular, new siRNA-LNPs designated as B49 showed a better toxicology profile when compared to the control B54 LNPs (Tab. 16).

    TABLE-US-00016 TABLE 16 Clinical biochemistry of plasma markers and hematology profile of new siRNA- LNPs designated as B49 compared to control siRNA-LNPs containing the benchmark lipid D-Lin-MC3-DMA (B54). Statistics were evaluated by Student's unpaired t-test. The p1 values are always relative to the control mice injected with PBS; the p2 values are always relative to the mice injected with control B54 LNPs; values of p < 0.01 are marked as c and p < 0.05 are marked as d. LNP B49 p1 p2 B54 p1 PBS Total bilirubin, 2.8 ? 1.06 2.5 ? 0.34 2.14 ? 0.43 ?mol/L AST, U/L 66 ? 12.06 .sup.150 ? 76.14 74.6 ? 7.78 ALT, U/L .sup.25 ? 3.79 75 ? 47.52 23.8 ? 1.6 HBDH, U/L 81.03 ? 18.8 d 148.7 ? 15.2 c 63.2 ? 8.24 LDH, U/L 310.1 ? 73.82 567 ? 61.8 c 231.2 ? 30.45 WBC, *10.sup.9/L 3.17 ? 0.91 6.58 ? 1.43 4.35 ? 0.44 Neutrophils, % 34 ? 7 d 51 ? 11 c 18 ? 2 Neutrophils, *10.sup.9/L 1.18 ? 0.55 3.65 ? 1.37 d 0.79 ? 0.09 Lymphocytes, % 64 ? 6 d 45 ? 1 C 77 ? 2 Lymphocytes, *10.sup.9/L 1.93 ? 0.41 2.69 ? 0.36 3.35 ? 0.38

    INDUSTRIAL APPLICABILITY

    [0280] The transfection particles containing lipidoids of the present invention are useful for a variety of biological applications in basic research, particularly cell culture or animal transfections to deliver active NA and subsequently silence or activate the chromosomal gene or genes, edit the genome or transcriptome, or allow the expression of protein encoded in the NA inserted using a transfection particle.

    [0281] In veterinary and human medicine, the transfection particles containing lipidoids of general formula I can preferably be used for therapeutic or prophylactic purposes. Therapeutic NA-containing particles can be administered to an animal or human to silence or activate chromosomal gene(s), silence or activate immunogens, inhibit or activate signaling pathways, edit the genome or transcriptome, or allow expression of the NA-encoded protein(s).

    [0282] The lipidoids of the formula I or transfection agents or transfection particles can also be used as medicaments, in particular for gene therapy, and are suitable for use in the treatment of malignancies and/or genetic disorders. They can also be formulated for cosmetic or biotechnological use.