Lipidoids for nucleic acid transfection and use thereof

Abstract

A lipidoid is disclosed of general formula I ##STR00001##
wherein X, Y, Z and R are as defined in the claims. This lipidoid is useful as a transfection agent. The disclosure further describes transfection agents, transfection particles containing this lipidoid, and their use.

Claims

1. Compounds of general formula I ##STR00009## wherein X is selected from a group consisting of 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), CC, CHCH, 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, NCH, and CHN; Y is independently selected from the group consisting of C.sub.2-C.sub.10 alkylene chains wherein in the alkylene chain, one or more CH.sub.2 groups may optionally be replaced with one or more 0 or S atoms; Z is selected from the group consisting of hydrogen, OH, CH.sub.2OH, 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, and NHCH.sub.3, N(CH.sub.3).sub.2, N.sup.+(CH.sub.3).sub.3, OCH.sub.3, OCH.sub.2CH.sub.3, C(O)R.sup.1, wherein R.sup.1 is selected from NH.sub.2, NH(CH.sub.2).sub.nOH, N[(CH.sub.2).sub.nOH].sub.2, NHCH(CH.sub.2OH).sub.2, NHCH.sub.2CH(OH)CH.sub.2OH, NH(CH.sub.2) C(O)NH.sub.2, N[CH.sub.2C(O)NH.sub.2].sub.2, NHCH[C(O)NH.sub.2].sub.2, NH(CH.sub.2).sub.2NHC(O)NH.sub.2, ##STR00010## wherein n is an integer within the range from 2 to 5; and R are the same or different from each other, each R being independently selected from the group consisting of C.sub.8-C.sub.20 alkyl, C.sub.8-C.sub.20 alkenyl, and C.sub.8-C.sub.20 alkynyl, wherein in the alkyl, alkenyl or alkynyl, one or more CH.sub.2 groups may optionally be replaced with one or more groups selected from CH(OH), OC(O), C(O)O, SS, C(O)NH, NHC(O), O, and S; and pharmaceutically acceptable salts, addition salts and solvates thereof.

2. The compound according to claim 1, wherein Z is selected from a group consisting of hydrogen, OH, CH.sub.2OH, NH.sub.2, N(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, and C(O)R.sup.1, wherein R.sup.1 is selected from NH.sub.2, NH(CH.sub.2).sub.nOH, N[(CH.sub.2).sub.nOH].sub.2, NHCH(CH.sub.2OH).sub.2, NHCH.sub.2CH(OH)CH.sub.2OH, NH(CH.sub.2).sub.nC(O)NH.sub.2, N[CH.sub.2C(O)NH.sub.2].sub.2, NHCH[C(O)NH.sub.2].sub.2, NH(CH.sub.2).sub.2NHC(O)NH.sub.2, ##STR00011## wherein n is an integer within the range from 2 to 5.

3. The compound according to claim 1, wherein X is selected from C(O)NH, a five membered heterocycle containing at least 2 nitrogen atoms, or C(O)O.

4. The compound according to claim 1, wherein R is independently selected from the group consisting of C.sub.8-C.sub.20 alkyl, C.sub.8-C.sub.20 alkenyl, and C.sub.8-C.sub.20 alkynyl, wherein in the said alkyl, alkenyl or alkynyl, one or more CH.sub.2 groups may optionally be replaced by one or more groups selected from CH(OH), OC(O), and C(O)O.

5. The compound according to claim 1, wherein all R in the molecule are the same, or all nitrogen atoms in the molecule are substituted identically by two identical R or two different R.

6. A transfection agent comprising at least one compound of general formula I according to claim 1 in an amount of 10 to 50 mol. %, and at least one helper lipid in a total amount of 50 to 90 mol. %.

7. A transfection agent comprising at least one compound of general formula I according to claim 1 in an amount of 15 to 30 mol. %, cholesterol in an amount of 30 to 55 mol. %, and at least one further helper lipid in an amount of 20 to 50 mol. %.

8. A transfection particle comprising at least one compound of general formula I according to claim 1, at least one nucleic acid and/or a part thereof and/or nucleic acid derivative, and at least one helper lipid.

9. A method of treatment comprising the step of administering the compound 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 to a subject in need thereof.

10. The method of treatment according to claim 9 for silencing or activating at least one chromosomal gene(s), silencing or activating immunogens, inhibiting or activating signaling pathways, editing genome or transcriptome, or enabling the expression of at least one protein(s) encoded by the nucleic acid.

11. The compound of general formula I according to claim 1 for transfecting cells or tissues with nucleic acid and/or a part thereof and/or nucleic acid derivative in vivo, preferably except for the transfection of human embryos for industrial or commercial purposes and except for the modification of a human germ line.

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

13. A method of administering a medicament, comprising the step of administering the compound of general formula I according to claim 1 for gene therapy.

14. A method of treatment of administering the compound of general formula I according to claim 1 in cosmetic preparations for delivering an active ingredient to the site of action to a subject in need thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1. Synthetic scheme of lipidoids 4a-g.

(2) FIG. 2. Synthetic scheme of lipidoid 9.

(3) FIG. 3. Synthetic scheme of lipidoid 13.

(4) FIG. 4. Synthetic scheme of compound 19, precursor of lipidoid 21.

(5) FIG. 5. Synthetic scheme of lipidoid 21.

(6) FIG. 6. Synthetic scheme of compound 22 and its acylchloride, precursors of lipidoids 23-25.

(7) FIG. 7. Synthetic scheme of lipidoids 23-25.

(8) FIG. 8. Synthetic scheme of lipidoids 29a-f

(9) FIG. 9. Synthetic scheme of lipidoids 31a-d.

(10) FIG. 10. Test of functional delivery of mRNA-LNP (B39) to murine liver.

EXAMPLES

List of Abbreviations

(11) eq. equivalent R.sub.f retention factor TLC thin-layer chromatography RVE rotary vacuum evaporator rt room temperature br s broad signal s singlet d doublet m multiplet dd doublet of doublets J interaction constant 6 chemical shift HRMS high-resolution mass spectrometry ESI electrospray ionization MALDI matrix-assisted laser desorption/ionisation IR infrared spectroscopy NMR nuclear magnetic resonance CE5 cyclohexane-ethylacetate mixture 95:5 (v/v) CE20 cyclohexane-ethylacetate mixture 80:20 (v/v) CE50 cyclohexane-ethylacetate mixture 50:50 (v/v) D1 dichloromethane-methanol-25% aqueous NH.sub.3 mixture 75:3 (v/v/v) D2 dichloromethane-methanol-25% aqueous NH.sub.3 mixture 175:22:3 (v/v/v) D3 dichloromethane-methanol-25% aqueous NH.sub.3 mixture 275:22:3 (v/v/v) D4 dichloromethane-methanol-25% aqueous NH.sub.3 mixture 375:22:3 (v/v/v) TFA trifluoroacetic acid HCTU O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate DIPEA N,N-diisopropylethylamine DMF N,N-dimethylformamide DCM dichloromethane ACN acetonitrile TBDPSCI tert-butyldiphenylchlorosilane DIC diisopropylcarbodiimide DMAP 4-dimethylaminopyridine LNP lipid nanoparticles NA nucleic acid DNA deoxyribonucleic acid RNA ribonucleic acid mRNA messenger RNA siRNA small interfering RNA tRNA transfer RNA miRNA micro RNA ssDNA/RNA single-stranded DNA/RNA dsDNA/RNA double-stranded DNA/RNA DMG-PEG.sub.2000 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine DOPE-Cy5 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5) Lip2000 Lipofectamine 2000 (Invitrogen)

Example 1

N.SUP.1.,N.SUP.1.-Didodecylethan-1,2-diamine 3a

(12) A 500 ml round-bottom flask equipped with a chlorocalcium cap and magnetic stirrer was filled with a solution of amine 1a (5.00 g, 31.2 mmol) in DCM (100 ml) and cooled to 0 C. in an ice bath. With intensive stirring, n-dodecylaldehyde (20.8 ml, 93.6 mmol, 3 eq.) was added, followed by sodium triacetoxyborohydride (19.8 g, 93.6 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. 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, 250 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 2a (5.12 g, 33.0%) was obtained as a yellowish oil.

(13) Trifluoroacetic acid (10 ml) was added to a solution of compound 2a (5.12 g) 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 11 separatory flask, diluted with 20% aqueous Na.sub.2CO.sub.3 (300 ml), and the product was extracted with DCM (250 ml, 250 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 3a (2.55 g, 62.4%; R.sub.f 0.46 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): =2.895, 2.64, 2.48, 1.45, 1.28, 1.26, 1.24-1.28, 1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =54.31, 53.85, 38.33, 31.90, 29.65, 29.62, 29.61, 29.52, 29.34, 26.36, 23.88, 22.67, 14.10 ppm. IR (film): .sub.max/cm.sup.1=3371 w and 3315 w ( NH.sub.2), 2801 m (.sub.s NCH.sub.2), 2953 s (.sub.asCH.sub.3), 2924 vs (.sub.as CH.sub.2), 2853 s (.sub.s CH.sub.2), 1467 m and 1457 m, sh (.sub.s 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.26H.sub.57N.sub.2 [M+H].sup.+ 397.45163; found 397.45093.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(2-(didodecylamino)ethyl) adamantane-1,3,5-tricarboxamide 4a

(14) O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU, 256 mg, 0.596 mmol, 4 eq.) and N,N-diisopropylethylamine (DIPEA, 0.415 ml, 2.39 mmol, 16 eq.) were added to a solution of adamantane-1,3,5-tricarboxylic acid (40 mg, 0.149 mmol) in anhydrous DMF (1.5 ml), and the solution was stirred for 15 min at room temperature. Then a solution of N.sup.1,N.sup.1-didodecylethane-1,2-diamine 3a (237 mg, 0.149 mmol, 4 eq.) in DCM (1.0 ml) was added, and the reaction mixture was stirred for 12 h. The solution was poured into a 250 ml separatory flask, diluted with saturated aqueous NaHCO.sub.3 (50 ml), and the product was extracted with DCM (50 ml, 220 ml). The combined organic phase was washed with brine (20 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 (20-50%). Lipidoid 4a (71 mg, 33.9%; R.sub.f 0.73 in D2 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained in the form of a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =7.33, 3.56, 3.32, 3.125, 3.075, 2.34, 2.01, 1.91, 1.79, 1.67, 1.36, 1.285, 1.26-1.32, 1.25, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =180.48, 56.72, 53.78, 41.38, 39.04, 36.86, 36.29, 31.90, 29.64, 29.62, 29.50, 29.40, 29.35, 29.04, 27.79, 26.36, 23.88, 22.68, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3440 w and 3322 w ( NH), 1653 w (amide I) and 1623 w (amide I bound), 1535 w (amide II), 2956 m (.sub.as CH.sub.3), 2927 vs (.sub.as CH.sub.2), 2855 m (.sub.s CH.sub.2), 1467 w and 1457 w (.sub.s CH.sub.2 and .sub.as CH.sub.3), 1378 w (.sub.s CH.sub.3). HRMS (MALDI): m/z calculated for C.sub.91H.sub.179N.sub.6O.sub.3 [M+H].sup.+ 1404.4033; found 1404.4012.

Example 2

N.SUP.1.,N.SUP.1.-Didodecylpropane-1,3-diamine 3b

(15) Amine 2b was prepared from amine 1b (6.0 g, 34.43 mmol), n-dodecylaldehyde (22.91 ml, 103.30 mmol, 3 eq.) and sodium triacetoxyborohydride (21.89 g, 103.30 mmol, 3 eq.) according to the procedure described for compound 2a in Example 1. Amine 2b was obtained as a yellowish oil (7.72 g, 43.9%).

(16) The deprotection of amine 2b was performed according to the procedure described for compound 2a in Example 1; diamine 3b (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): .sub.max/cm.sup.1=3361 w and 3274 w ( NH.sub.2), 2803 m (.sub.s NCH.sub.2), 2954 s (.sub.asCH.sub.3), 2924 vs (.sub.as CH.sub.2), 2853 s (.sub.s CH.sub.2), 1467 m and 1456 m, sh (.sub.s CH.sub.2 and .sub.as CH.sub.3), 1378 w and 1364 w (.sub.s CH.sub.3), 720 m (.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.

N.SUP.1.,N.SUP.3.,N.SUP.5.-tris(3-(didodecylamino)propyl)adamantane-1,3,5-tricarboxamide 4b

(17) Lipidoid 4b was prepared from adamantane-1,3,5-tricarboxylic acid (20 mg, 0.075 mmol), HCTU (128 mg, 0.298 mmol, 4 eq.), DIPEA (0.208 ml, 1.19 mmol, 16 eq.) and diamine 3b (123 mg, 0.298 mmol, 4 eq.) according to the procedure described for compound 4a in Example 1; lipidoid 4b (64 mg, 59.3%; R.sub.f 0.51 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained as a viscous yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =7.59, 3.36, 2.99, 2.88, 2.31, 2.13, 2.00, 1.96, 1.81, 1.67, 1.31, 1.28, 1.25-1.30, 1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =177.39, 55.22, 50.70, 41.78, 40.38, 37.20, 35.67, 31.89, 29.60, 29.51, 29.48, 29.32, 29.19, 28.39, 26.94, 24.10, 23.68, 22.67, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3466 w and 3287 w ( NH), 1656 m (amide I) and 1511 m (amide II), 2814 w (.sub.s CH.sub.2NR.sub.2), 2954 s (.sub.as CH.sub.3), 2927 vs (.sub.as CH.sub.2), 2871 m (.sub.s CH.sub.3), 2855 s (.sub.sCH.sub.2), 1468 m and 1456 m (.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). HRMS (MALDI): m/z calculated for C.sub.94H.sub.185O.sub.3N.sub.6 [M+H].sup.+ 1446.45027; found 1446.44896.

Example 3

N.SUP.1.,N.SUP.1.-Didodecylbutane-1,4-diamine 3c

(18) Amine 2c was prepared from amine 1c (5.0 g, 26.56 mmol), n-dodecylaldehyde (17.67 ml, 79.67 mmol, 3 eq.) and sodium triacetoxyborohydride (16.89 g, 79.67 mmol, 3 eq.) according to the procedure described for compound 2a in Example 1. Amine 2c was obtained as a yellowish oil (4.15 g, 29.8%). The deprotection of amine 2c was performed according to the procedure described for compound 2a in Example 1; diamine 3c (2.36 g, 70.3%; R.sub.f 0.29 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): =2.85, 2.81, 2.59, 1.725, 1.68, 1.51, 1.28, 1.265, 1.25-1.30, 1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =53.33, 52.74, 40.44, 31.90, 29.64, 29.60, 29.60, 29.40, 29.33, 28.65, 27.46, 24.83, 24.40, 22.67, 14.10 ppm. IR (film): .sub.max/cm.sup.1=3370 w and 3274 w ( NH.sub.2), 2798 m (.sub.s NCH.sub.2), 2957 s (.sub.as CH.sub.3), 2924 vs (.sub.as CH.sub.2), 2853 s (.sub.s CH.sub.2), 1467 m and 1456 m, sh (.sub.s CH.sub.2 and .sub.as CH.sub.3), 1378 w and 1367 w (.sub.s CH.sub.3), 720 m (.sub.as CH.sub.2). HRMS (ESI): m/z calculated for C.sub.28H.sub.61N.sub.2 [M+H].sup.+ 425.48293; found 425.48227.

N.SUP.1.,N.SUP.3.,N.SUP.5.-tris(4-(didodecylamino)butyl)adamantane-1,3,5-tricarboxamide 4c

(19) Lipidoid 4c was prepared from adamantane-1,3,5-tricarboxylic acid (40 mg, 0.149 mmol), HCTU (256 mg, 0.596 mmol, 4 eq.), DIPEA (0.416 ml, 2.39 mmol, 16 eq.) and diamine 3c (253 mg, 0.596 mmol, 4 eq.) according to the procedure described for compound 4a in Example 1; lipidoid 4c (64 mg, 28.8%; R.sub.f 0.48 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained in the form of a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =7.27, 3.335, 3.03, 2.98, 2.28, 2.12, 2.06, 1.875, 1.81, 1.74, 1.64, 1.33, 1.28, 1.25-1.29, 1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =177.27, 52.78, 52.27, 41.83, 40.26, 37.36, 37.20, 31.88, 29.58, 29.49, 29.43, 29.31, 29.10, 28.37, 26.82, 26.56, 22.94, 22.66, 20.79, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3441 w and 3329 w ( NH), 1641 m (amide I), 1534 w (amide II), 2956 m (.sub.as CH.sub.3), 2927 vs (.sub.as CH.sub.2), 2855 m (.sub.s CH.sub.2), 1466 m and 1458 m (.sub.s CH.sub.2 and .sub.as CH.sub.3), 1378 w (.sub.s CH.sub.3). HRMS (MALDI): m/z calculated for C.sub.97H.sub.191N.sub.6O.sub.3 [M+H].sup.+ 1488.4972; found 1488.4956.

Example 4

N.SUP.1.,N.SUP.1.-Didodecylpentane-1,5-diamine 3d

(20) Amine 2d was prepared from amine 1d (5.0 g, 24.72 mmol), n-dodecylaldehyde (16.45 ml, 74.15 mmol, 3 eq.) and sodium triacetoxyborohydride (15.71 g, 74.15 mmol, 3 eq.) According to the procedure described for compound 2a in Example 1. Amine 2d was obtained as a yellowish oil (6.01 g, 45.1%).

(21) The deprotection of amine 2d was performed according to the procedure described for compound 2a in Example 1; diamine 3d (4.32 g, 88.3%; R.sub.f 0.28 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained as a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =2.75, 2.68, 2.55, 1.565, 1.53, 1.50, 1.35, 1.28, 1.27, 1.24-1.28, 1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =53.61, 53.55, 41.48, 32.10, 31.89, 29.63, 29.61, 29.58, 29.45, 29.32, 27.41, 25.74, 24.90, 24.55, 22.66, 14.09 ppm. IR (film): .sub.max/cm.sup.1=3367 w and 3284 w ( NH.sub.2), 2797 m (vs NCH.sub.2), 2956 s (vas CH.sub.3), 2924 vs (.sub.as CH.sub.2), 2853 s (.sub.s CH.sub.2), 1467 m and 1456 m, sh (.sub.s CH.sub.2 and .sub.as CH.sub.3), 1378 w and 1367 w (.sub.s CH.sub.3), 720 m (.sub.as CH.sub.2). HRMS (ESI): m/z calculated for C.sub.29H.sub.63N.sub.2 [M+H].sup.+ 439.49858; found 439.49783.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(5-(didodecylamino)pentyl)adamantane-1,3,5-tricarboxamide 4d

(22) Lipidoid 4d was prepared from adamantane-1,3,5-tricarboxylic acid (40 mg, 0.149 mmol), HCTU (256 mg, 0.596 mmol, 4 eq.), DIPEA (0.416 ml, 2.39 mmol, 16 eq.) and diamine 3d (262 mg, 0.596 mmol, 4 eq.) according to the procedure described for compound 4a in Example 1; lipidoid 4d (74 mg, 32.4%; R.sub.f 0.49 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained as a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =6.91, 3.27, 2.99, 2.29, 2.09, 1.97, 1.82, 1.81, 1.76, 1.59, 1.45, 1.34, 1.28, 1.25-1.30, 1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =177.01, 52.77, 52.30, 41.82, 40.10, 38.33, 37.59, 31.88, 29.58, 29.48, 29.43, 29.30, 29.09, 28.53, 28.39, 26.84, 23.83, 23.09, 23.02, 22.66, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3322 w ( NH), 1640 m (amide I), 1535 w (amide II), 2956 m (.sub.as CH.sub.3), 2927 vs (.sub.as CH.sub.2), 2855 m (.sub.s CH.sub.2), 1467 m and 1457 m (.sub.s CH.sub.2 and .sub.as CH.sub.3), 1378 w (.sub.s CH.sub.3), 722 m (.sub.as CH.sub.2). HRMS (MALDI): m/z calculated for C.sub.100H.sub.197N.sub.6O.sub.3 [M+H].sup.+1530.5442; found 1530.5478.

Example 5

N.SUP.1.,N.SUP.1.-Didodecylhexane-1,6-diamine 3e

(23) Amine 2e was prepared from amine 1e (5.0 g, 23.11 mmol), n-dodecylaldehyde (15.38 ml, 69.34 mmol, 3 eq.) and sodium triacetoxyborohydride (14.70 g, 69.34 mmol, 3 eq.) according to the procedure described for compound 2a in Example 1. Amine 2e was obtained as a yellowish oil (3.67 g, 28.7%).

(24) The deprotection of amine 2e was performed according to the procedure described for compound 2a in Example 1; diamine 3e (2.17 g, 72.2%; R.sub.f 0.31 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained as a yellowish oil. .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): .sub.max/cm.sup.1=3374 w and 3294 w ( NH.sub.2), 2797 m (.sub.s NCH.sub.2), 2956 s (.sub.a CH.sub.3), 2924 vs (.sub.as CH.sub.2), 2853 s (.sub.s CH.sub.2), 1467 m and 1455 m, sh (.sub.s 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.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(didodecylamino)hexyl)adamantane-1,3,5-tricarboxamide 4e

(25) Lipidoid 4e was prepared from adamantane-1,3,5-tricarboxylic acid (40 mg, 0.149 mmol), HCTU (256 mg, 0.596 mmol, 4 eq.), DIPEA (0.416 ml, 2.39 mmol, 16 eq.) and diamine 3e (270 mg, 0.596 mmol, 4 eq.) according to the procedure described for compound 4a in Example 1; lipidoid 4e (91 mg, 38.8%; R.sub.f 0.52 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained as a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =7.80, 3.33, 3.03, 2.99, 2.37, 2.27, 2.02, 1.92, 1.81, 1.76, 1.62, 1.43, 1.41, 1.34, 1.285, 1.26-1.30, 1.24, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =177.87, 52.87, 52.26, 41.74, 39.31, 36.86, 31.90, 29.60, 29.50, 29.44, 29.32, 29.10, 28.26, 26.82, 25.77, 25.52, 23.44, 23.14, 22.68, 14.12 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3463 w and 3327 w ( NH), 1641 m (amide I), 1535 m (amide II), 2958 s (.sub.as CH.sub.3), 2871 s (.sub.s CH.sub.3), 1467 m and 1457 m (.sub.s CH.sub.2 and .sub.as CH.sub.3), 2927 s (.sub.as CH.sub.2), 2855 s (.sub.s CH.sub.2), 1378 w (.sub.s CH.sub.3), 721 w (.sub.as CH.sub.2), 2799 w (.sub.s CH.sub.2NR.sub.2). HRMS (MALDI): m/z calculated for C.sub.103H.sub.203N.sub.6O.sub.3 [M+H].sup.+ 1572.5911; found 1572.5881.

Example 6

N.SUP.1.,N.SUP.1.-Didodecylheptane-1,7-diamine 3f

(26) Amine 2f was prepared from amine 1f (1.0 g, 4.34 mmol), n-dodecylaldehyde (3.14 ml, 13.02 mmol, 3 eq.) and sodium triacetoxyborohydride (2.76 g, 13.02 mmol, 3 eq.) according to the procedure described for compound 2a in Example 1. Amine 2f was obtained as a yellowish oil (1.74 g, 70.6%).

(27) The deprotection of amine 2f was performed in a mixture of TFA (4 ml) and DCM (4 ml) according to the procedure described for 2a in Example 1; diamine 3f (0.842 g, 59.1%; R.sub.f 0.38 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained as a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =2.73, 2.69, 2.64, 1.56, 1.50, 1.30, 1.28, 1.25-1.31, 1.25, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =53.37, 53.20, 41.62, 32.22, 31.89, 27.10-29.60, 26.57, 25.14, 22.66, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3391 vw (.sub.as NH.sub.2); 2960 s, sh (.sub.a CH.sub.3); 2927 vs (.sub.as CH.sub.2); 2872 s, sh (.sub.s CH.sub.3); 2855 vs (.sub.s CH.sub.2); 2798 m (.sub.s NCH.sub.2); 1467 s and 1458 m (.sub.s CH.sub.2 and .sub.as CH.sub.3); 1378 w (.sub.sCH.sub.3); 1302 w (.sub.s CH.sub.2); 721 w (.sub.as and .sub.as CH.sub.2). HRMS (ESI): m/z calculated for C.sub.31H.sub.67N.sub.2 [M+H].sup.+ 467.52988; found 467.52974.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(7-(didodecylamino)heptyl)adamantane-1,3,5-tricarboxamide 4f

(28) Lipidoid 4f was prepared from adamantane-1,3,5-tricarboxylic acid (40 mg, 0.149 mmol), HCTU (256 mg, 0.596 mmol, 4 eq.), DIPEA (0.416 ml, 2.39 mmol, 16 eq.) and diamine 3f (278 mg, 0.596 mmol, 4 eq.) according to the procedure described for compound 4a in Example 1; lipidoid 4f (199 mg, 82.6%; R.sub.f 0.55 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained as a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =7.97, 3.22, 3.09, 2.34, 1.92, 1.83, 1.82, 1.70, 1.58, 1.52, 1.36, 1.30, 1.285, 1.28, 1.25-1.32, 1.25, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =177.27, 54.00, 53.14, 41.59, 39.48, 39.30, 36.92, 31.89, 29.60, 29.48, 29.39, 29.32, 29.06, 28.22, 26.50, 25.58, 23.63, 23.38, 22.67, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3438 w (free) and 3339, 3196 w (bound) ( NH); 1627 m (amide I); 1535 m (amide II); 2956 s, sh (.sub.as CH.sub.3); 2873 m, sh (.sub.s CH.sub.3); 1468 m and 1457 m, sh (.sub.s CH, and .sub.a, CH.sub.3); 2927 vs (.sub.as CH.sub.2); 2856 s (.sub.s CH.sub.2); 1378 w (.sub.s CH.sub.3); 722 w (.sub.as CH.sub.2); 2805 w ( CH.sub.2NR.sub.2). HRMS (MALDI): m/z calculated for C.sub.106H.sub.209N.sub.6O.sub.3 [M+H].sup.+ 1614.6381; found 1614.6414.

Example 7

N.SUP.1.,N.SUP.1.-Didodecyloctane-1,8-diamine 3g

(29) Amine 2g was prepared from amine 1g (1.0 g, 4.09 mmol), n-dodecylaldehyde (2.96 ml, 12.28 mmol, 3 eq.) and sodium triacetoxyborohydride (2.60 g, 12.28 mmol, 3 eq.) according to the procedure described for compound 2a in Example 1. The amine 2g was obtained as a yellowish oil (1.98 g, 83.1%).

(30) The deprotection of amine 2g was performed in a mixture of TFA (4 ml) and DCM (4 ml) according to the procedure described for 2a in Example 1; diamine 3g (0.848 g, 52.0%; 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): =2.70, 2.63, 2.53, 1.51, 1.47, 1.32, 1.28, 1.25-1.32, 1.25, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =53.61, 53.20, 41.92, 33.00, 31.90, 27.30-29.60, 26.70, 25.83, 22.67, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3391 vw (.sub.as NH.sub.2); 2960 s, sh (.sub.as CH.sub.3); 2927 vs (.sub.asCH.sub.2); 2872 s, sh (.sub.s CH.sub.3); 2855 vs (.sub.s CH.sub.2); 2799 m (.sub.s NCH.sub.2); 1467 s and 1458 m (.sub.s CH.sub.2 and .sub.asCH.sub.3); 1378 w (.sub.s CH.sub.3); 1302 w (.sub.s CH.sub.2); 721 w (.sub.as and .sub.as CH.sub.2). HRMS (ESI): m/z calculated for C.sub.32H.sub.69N.sub.2 [M+H].sup.+ 481.54553; found 481.54507.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(8-(didodecylamino)octyl)adamantane-1,3,5-tricarboxamide 4g

(31) Lipidoid 4g was prepared from adamantane-1,3,5-tricarboxylic acid (40 mg, 0.149 mmol), HCTU (256 mg, 0.596 mmol, 4 eq.), DIPEA (0.416 ml, 2.39 mmol, 16 eq.) and diamine 3g (286 mg, 0.596 mmol, 4 eq.) according to the procedure described for compound 4a in Example 1; lipidoid 4g (211 mg, 85.4%; R.sub.f 0.60 in mobile phase D2 on a TLC plate pre-saturated with ammonia, detection with ninhydrin) was obtained as a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =8.03, 3.21, 3.09, 3.07, 2.35, 1.985, 1.82, 1.815, 1.71, 1.51, 1.335, 1.33, 1.29, 1.28, 1.25-1.33, 1.25, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =177.22, 54.04, 53.07, 41.56, 40.01, 39.08, 36.95, 31.89, 29.60, 29.49, 29.40, 29.32, 29.06, 28.45, 28.42, 28.30, 26.53, 26.13, 26.01, 23.88, 23.45, 22.67, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3439 w (free) and 3341, 3196 w (bound) ( NH); 1635, 1627 w (amide I); 1533 w (amide II); 2954 m, sh (.sub.asCH.sub.3); 2873 m, sh (.sub.s CH.sub.3); 1467 m and 1457 w, sh (.sub.s CH.sub.2 and .sub.as CH.sub.3); 2927 vs (.sub.as CH.sub.2); 2856 s (.sub.s CH.sub.2); 1378 w (.sub.s CH.sub.3); 2810 vw, sh (.sub.s CH.sub.2NR.sub.2). HRMS: m/z calculated for C.sub.109H.sub.215N.sub.6O.sub.3 [M+H].sup.+ 1656.6856; found 1656.6882.

Example 8

1,2-Epoxydodecane 6

(32) N-chlorosuccinimide (NCS, 3.44 g, 25.77 mmol, 0.95 eq.) and L-proline (0.937 g, 8.14 mmol, 0.30 eq.) were added to a solution of n-dodecylaldehyde (6.0 ml, 27.13 mmol) in acetonitrile (70 ml), cooled to 0 C. in an ice bath, and the mixture was stirred at 0 C. for 2 h. Then the solution was diluted with ethanol (40 ml), NaBH.sub.4 (2.57 g, 67.82 mmol, 2.5 eq.) was added, and the reaction mixture was stirred at 0 C. for 3.5 h. The solution was poured into a 1000 ml separatory flask, diluted with water (100 ml) and brine (100 ml), and the product was extracted with ethyl acetate (300 ml, 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 ethyl acetate in cyclohexane (0-20%). Chloroalcohol 5 (2.79 g, 46.6%; R.sub.f 0.42 in mobile phase CE20, detection with KMnO.sub.4) was obtained as a colorless oil.

(33) A solution of NaOH (11.37 g, 0.284 mmol, 22.5 eq.) in water (49 ml) was added to a solution of chloroalcohol 5 (2.79 g, 12.64 mmol) in dioxane (38 ml), and the mixture was stirred for 30 h at 35 C. The solution was then poured into a 500 ml separatory flask, diluted with water (100 ml), and the product was extracted with DCM (100 ml, 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 crude product was purified by silica gel column chromatography using a linear gradient of ethyl acetate in cyclohexane (0-5%). Epoxide 6 (1.797 g, 77.2%; R.sub.f 0.38 in mobile phase CE5, detection with phosphomolybdic acid/Ce.sup.4+) was obtained as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3): =0.92 (t, J=6 Hz, 3H), 1.29-1.60 (m, 18H), 2.47-2.49 (m, 1H), 2.76-2.78 (m, 1H), 2.90-2.95 (m, 1H) ppm. .sup.13C NMR (101 MHz, CDCl.sub.3) =14.11, 22.68, 25.97, 29.33, 29.45, 29.56, 29.59, 31.90, 32.50, 47.14, 52.42 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=epoxid: 2997 w, sh (.sub.as CH.sub.2); 1482 w, 1410 w, 1130 w (.sub.s OCH.sub.2); 1259 w (.sub.s skeleton, respiratory); 917 w (.sub.as circle); 896 vw (.sub.as COC); alif. chain: 2957 s (.sub.as CH.sub.3); 2928 vs (.sub.as CH.sub.2); 2872 m (.sub.s CH.sub.3); 2856 s (.sub.s CH.sub.2); 1467 m and 1458 m (.sub.s CH.sub.2 and .sub.asCH.sub.3); 1379 w (.sub.s CH.sub.3), HRMS (EI): m/z calculated for C.sub.12H.sub.24O [M].sup.+ 184.1827; found 184.1832.

N.SUP.1.,N.SUP.1.-Bis(2-hydroxydodecyl)hexan-1,6-diamine 8

(34) Amine 1e (0.86 g, 3.98 mmol) and epoxide 6 (1.76 g, 9.54 mmol, 2.4 eq.) were mixed in a 4 ml glass vial, and the mixture was heated in the absence of solvent to 80 C. under an argon atmosphere for 24 h. The resulting yellowish liquid was purified by silica gel column chromatography using a linear gradient of D1 in DCM (0-30%). Amine 7 (1.98 g, 85.1%; R.sub.f 0.51 in mobile phase D3, detection with ninhydrin) was obtained as a yellowish oil.

(35) The deprotection of amine 7 was performed in a mixture of TFA (4 ml) and DCM (6 ml) according to the procedure described for compound 2a in Example 1; diamine 8 (1.271 g, 77.4%; R.sub.f 0.20 in mobile phase D2, detection with ninhydrin) was obtained as a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =3.65, 3.63, 2.84, 2.82, 2.565, 2.55, 2.41, 2.325, 1.60, 1.59, 1.41, 1.38, 1.35, 1.30-1.48, 1.28, 1.25-1.29, 1.25, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =69.39, 67.71, 62.72, 61.05, 55.78, 54.77, 40.86, 40.56, 35.22, 35.08, 31.90, 30.45, 29.6-29.9, 29.33, 25.65-26.77, 22.67, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3412 w, br ( OH); 1077 w, br ( COH); 1621 vw, br (.sub.s NH.sub.2); 1090 w ( CNH.sub.2); 2956 m, sh (.sub.as CH.sub.3); 2928 vs (.sub.as CH.sub.2); 2871 m (.sub.s CH.sub.3); 2855 s (.sub.s CH.sub.2); 2810 w, sh (.sub.s NCH.sub.2); 1467 w and 1457 w (.sub.s CH.sub.2 and .sub.as CH.sub.3); 1378 vw (.sub.s CH.sub.3); 722 vw (.sub.as and .sub.as CH.sub.2). HRMS (ESI): m/z calculated for C.sub.30H.sub.65N.sub.2O.sub.2 [M+H].sup.+ 485.50406; found 485.50461.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(bis(2-hydroxydodecyl)amino)hexyl)adamantane-1,3,5-tricarboxamide 9

(36) Lipidoid 9 was prepared from adamantane-1,3,5-tricarboxylic acid (40 mg, 0.149 mmol), HCTU (256 mg, 0.596 mmol, 4 eq.), DIPEA (0.416 ml, 2.39 mmol, 16 eq.) and diamine 8 (289 mg, 0.596 mmol, 4 eq.) according to the procedure described for compound 4a in Example 1; lipidoid 9 (188 mg, 75.5%; R.sub.f 0.43 in mobile phase D2, detection with ninhydrin) was obtained as a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =6.97, 4.10, 4.075, 4.04, 4.00, 3.38, 3.31, 3.28, 3.24, 3.21, 3.19, 3.16, 3.11, 2.34, 2.14, 1.93, 1.87, 1.28, 1.25-1.31, 1.25, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =177.50, 66.38, 65.92, 65.10, 64.73, 61.43, 61.23, 60.55, 59.64, 57.63, 54.48, 53.38, 41.70, 39.42, 39.23, 37.16, 31.90, 29.63, 29.56, 29.52, 29.34, 28.39, 22.68, 14.11 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3300-3500 m, br ( OH); 1090 w ( COH); 3439 w (free) and 3344 w (bound) ( NH); 1635 m (amide I); 1536 m (amide II); 2956 m, sh (.sub.as CH.sub.3); 2873 m, sh (.sub.s CH.sub.3); 2927 vs (.sub.as CH.sub.2); 2855 s (.sub.s CH.sub.2); 1378 w (.sub.s CH.sub.3); 2808 w, sh (.sub.s CH.sub.2NR.sub.2); 721 w (.sub.s CH.sub.2). HRMS (MALDI): m/z calculated for C.sub.103H.sub.203N.sub.6O.sub.9 [M+H].sup.+ 1668.5612; found 1668.5628.

Example 9

Linoleylaldehyde 10

(37) Dess-Martin periodinane (4.45 g, 10.49 mmol, 1.3 eq.) was added to a solution of linoleyl alcohol (2.50 ml, 8.07 mmol) in DCM (120 ml), cooled to 0 C. in an ice bath, and the mixture was stirred at 0 C. for 4 h. The reaction was then quenched by the addition of sodium thiosulphate solution (20 g Na.sub.2S.sub.2O.sub.3.Math.5H.sub.2O/100 ml H.sub.2O) and saturated aqueous sodium bicarbonate solution (50 ml), and stirred for 1 h at rt 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, 250 ml). The combined organic phase was washed with brine (150 ml), dried over anhydrous sodium sulphate, filtered through an S2 frit, and the solvents were evaporated in an RVE. The crude material was purified by silica gel column chromatography (isocratic conditions, 5% ethyl acetate in cyclohexane). Aldehyde 10 (1.271 g, 59.6%; R.sub.f 0.36 in mobile phase CE5, detection with KMnO4) 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 12

(38) Amine 11 was prepared from amine 1e (0.345 g, 1.59 mmol), aldehyde 10 (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 2a in Example 1. Amine 11 was obtained as a yellowish oil (1.08 g, 94.9%; R.sub.f 0.18 in mobile phase CE20, detection with ninhydrin).

(39) The deprotection of amine 11 was performed in a mixture of TFA (4 ml) and DCM (5 ml) according to the procedure described for 2a in Example 1; diamine 12 (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): .sub.max/cm.sup.1=3011 s (.sub.as=CH); 1646-1673 m ( CC); 3455 w (.sub.as NH.sub.2); 3394 (.sub.s NH.sub.2); 1620 w (.sub.s NH.sub.2); 1087 m ( CNH.sub.2); 2957 s, sh (.sub.as CH.sub.3); 2928 vs (.sub.as CH.sub.2); 2873 s, sh (.sub.s CH.sub.3); 2856 vs (.sub.s CH.sub.2); 2801 m (.sub.s NCH.sub.2); 1467 m and 1457 m, sh (.sub.s 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.1N.sub.2 [M+H].sup.+ 613.63943; found 613.63899.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(bis((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)hexyl)adamantane-1,3,5-tricarboxamide 13

(40) Lipidoid 13 was prepared from adamantane-1,3,5-tricarboxylic acid (30 mg, 0.112 mmol), HCTU (192 mg, 0.447 mmol, 4 eq.), DIPEA (0.312 ml, 2.39 mmol, 16 eq.) and diamine 12 (274 mg, 0.447 mmol, 4 eq.) according to the procedure described for compound 4a in Example 1; lipidoid 13 (154 mg, 67.1%; R.sub.f 0.48 in mobile phase D2, detection with ninhydrin) was obtained as a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =6.32, 5.37, 5.32, 3.23, 2.76, 2.31, 2.04, 1.90, 1.81, 1.52, 1.35, 1.305, 1.295, 1.29, 1.28-1.35, 1.28, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.42, 130.21, 129.99, 128.04, 127.87, 52.94, 41.72, 39.98, 38.99, 37.77, 31.50, 29.60, 29.18, 29.08, 29.0-29.7, 28.57, 27.18, 26.26, 25.61, 22.55, 14.07 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3011 m (.sub.as=CH); 1661 m ( CC); 3464 w (free) and 3347 w (bound) ( NH); 1645 m, sh (amide I); 1534 w (amide II); 2957 m, sh (.sub.as CH.sub.3); 2873 m, sh (.sub.s CH.sub.3); 2929 vs (.sub.as CH.sub.2); 2856 s (.sub.s CH.sub.2); 1378 and 1366 w (.sub.s CH.sub.3); 1086 w, sh ( CN); 722 w (.sub.as and .sub.as CH.sub.2). HRMS (MALDI): m/z calculated for C.sub.139H.sub.251N.sub.6O.sub.3 [M+H].sup.+ 2052.9667; found 2052.9672.

Example 10

8-((tert-Butyldiphenylsilyl)oxy)octane-1-ol 14

(41) Tert-butyldiphenylchlorosilane (17.50 ml, 68.39 mmol, 1 eq.) was added to a solution of 1,8-octanediol (10.0 g, 68.39 mmol) and imidazole (5.59 g, 82.06 mmol, 1.2 eq.) in DCM (250 ml), and the reaction mixture was stirred for 24 h at rt. The solution was poured into a 1000 ml separatory flask, diluted with water (400 ml) and brine (100 ml), and the product was extracted with DCM (2100 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 ethyl acetate in cyclohexane (0-30%). Alcohol 14 (13.65 g, 51.9%; R.sub.f 0.35 in mobile phase CE20, detection with KMnO.sub.4) was obtained as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3): =1.08 (s, 9H), 1.31-1.41 (m, 8H), 1.55-1.62 (m, 4H), 3.64-3.70 (m, 4H), 7.38-7.47 (m, 6H), 7.69-7.71 (m, 4H) ppm. .sup.13C NMR (101 MHz, CDCl.sub.3): =19.24, 25.68, 25.72, 26.89, 29.33, 29.38, 32.56, 32.79, 63.09, 63.99, 127.57, 129.48, 134.19, 135.58 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3072 m (20a); 3053 m (20b); 2529 s, sh (tBu, .sub.as CH.sub.3); 2898 s, sh (tBu, .sub.s CH.sub.3); 1590, 1568 (8a, 8b); 1487 m (19a); 1463 m, 1473 s (tBu, .sub.as CH.sub.3); 1428 s (19b); 1390 m, 1362 m (tBu, .sub.s CH.sub.3); 1189 m; 1112 vs, 1094 vs (.sub.as Si-Ph); 1030 m (18a); 1008 m ( Ph-Si); 939 m (r CH.sub.3); 701 vs (.sub.s COSi); 688 m (4); 622 m (6b); 614 s (6a); 505 s (16b); 489 m ( Si-Ph); 2932 vs (.sub.as CH.sub.2); 2858 vs (.sub.s CH.sub.2); 3636 m, 3341 m, br (.sub.s OH); 1057 m (.sub.s COH). HRMS (ESI): m/z calculated for C.sub.24H.sub.36O.sub.2NaSi [M+Na].sup.+ 407.23768; found 407.23742.

8-((tert-Butyldiphenylsilyl)oxy)octanoic acid 15

(42) Dess-Martin periodinane (19.54 g, 46.07 mmol, 1.3 eq.) was added to a solution of alcohol 14 (13.63 g, 35.44 mmol) in DCM (250 ml), cooled to 0 C. in an ice bath, and the mixture was stirred at 0 C. for 4 h. The reaction was then quenched by the addition of sodium thiosulphate solution (50 g Na.sub.2S.sub.2O.sub.3.Math.5H.sub.2O/150 ml H.sub.2O) and saturated aqueous sodium bicarbonate solution (100 ml), and stirred for 1 h at rt until the initially milky solution turned clear. The solution was poured into a 1000 ml separatory flask, diluted with water (200 ml), and the product was extracted with DCM (200 ml, 250 ml). The combined organic phase was washed with brine (200 ml), dried over anhydrous sodium sulphate, filtered through an S2 frit, and the solvents were evaporated in an RVE. 13.56 g of a colourless oil were obtained.

(43) The obtained residue was dissolved in a 2 l flask in a mixture of acetone (450 ml) and water (90 ml); 2-methyl-2-butene (15.02 ml, 141.8 mmol, 4 eq.) and NaH.sub.2PO.sub.4.Math.2H.sub.2O (11.06 g, 70.88 mmol, 2 eq.) were added to the solution, and the suspension was cooled to 0 C. in an ice bath. A solution of sodium chlorite (9.62 g, 106.32 mmol, 3 eq.) in water (60 ml) was then added gradually from a dropping funnel over 30 min, the reaction mixture was removed from the cooling bath, and stirred vigorously at rt for 12 h. The solution was poured into a 1000 ml separatory flask, diluted with a solution of citric acid (70 g) in water (300 ml), and the product was extracted with diethyl ether (300 ml, 50 ml). The combined organic phase was washed with brine (300 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 (isocratic conditions, 5% methanol in chloroform). Acid 15 (12.92 g, 91.5%; R.sub.f 0.35 in mobile phase MeOHCHCl.sub.3 5:95 (v/v), detection with KMnO.sub.4) was obtained as a viscous colourless oil. .sup.1H NMR (400 MHz, CDCl.sub.3): =1.05 (s, 9H), 1.27-1.39 (m, 6H), 1.51-1.66 (m, 4H), 2.34 (t, J=7.5 Hz, 2H), 3.65 (t, J=6.5 Hz, 2H), 7.36-7.44 (m, 6H), 7.66-7.68 (m, 4H) ppm. .sup.13C NMR (101 MHz, CDCl.sub.3): =19.23, 24.63, 25.59, 26.89, 28.97, 29.02, 32.48, 33.95, 63.91, 127.57, 129.49, 134.15, 135.58, 179.60 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3534 w ( OH, monomer); 3100 w, 2740 w, 2674 w ( OH, dimer); 1711 vs ( CO); 1413 w, 1289 w ( CO and COH); 2561 m, sh (tBu, .sub.as CH.sub.3); 2898 m, sh (tBu, .sub.s CH.sub.3); 1590 w; 1487 w (19a); 1463 m, 1472 m (tBu, .sub.as CH.sub.3); 1429 m (19b); 1390 w, 1362 w (tBu, .sub.s CH.sub.3); 1112 s, (.sub.as COSi); 1093 m (18b); 1030 w; 1008 w ( Ph-Si); 940 w (r CH.sub.3); 701 s (4); 688 w (SiOC); 622 w (6b); 614 m (6a); 505 w (16b); 2932 s (.sub.as CH.sub.2); 2858 m (.sub.s CH.sub.2). HRMS (ESI): m/z calculated for C.sub.24H.sub.33O.sub.3Si [M+H].sup.+ 397.22044; found 397.22018.

(Z)-Non-2-en-1-yl-8-hydroxyoctanoate 17

(44) Diisopropylcarbodiimide (1.08 ml, 6.90 mmol, 1.1 eq.) and 4-dimethylaminopyridine (23.0 mg, 0.188 mmol, 0.03 eq.) were added to a solution of acid 15 (2.50 g, 6.27 mmol) in DCM (100 ml), cooled to 0 C. in an ice bath, and the mixture was stirred at 0 C. for 30 min. Then trans-2-nonen-1-ol (1.37 ml, 8.15 mmol, 1.3 eq.) was added, and the reaction mixture was stirred for 12 h at rt. The solvent was evaporated in an RVE, and the residue was purified by silica gel column chromatography (isocratic conditions, 5% ethyl acetate in cyclohexane). Ester 16 (2.784 g, 84.9%; R.sub.f 0.61 in mobile phase CE5, detection with KMnO.sub.4) was obtained as a colorless oil.

(45) A solution of tetrabutylammonium fluoride monohydrate (2.95 g, 10.56 mmol, 2 eq.) in tetrahydrofuran (10 ml), was added to a solution of ester 16 (2.76 g, 5.28 mmol) in tetrahydrofuran (40 ml), and the reaction the mixture was stirred for 20 h at rt. The solution was poured into a 500 ml separatory flask, diluted with 10% aqueous ammonium chloride solution (150 ml), and the product was extracted with diethyl ether (150 ml, 50 ml). The combined organic phase was washed with brine (100 ml), dried over anhydrous sodium sulfate, 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 ethyl acetate in cyclohexane (0-45%). Alcohol 17 (1.311 g, 87.3%; R.sub.f 0.61 in mobile phase CE50, detection with KMnO.sub.4) was obtained as a slightly yellowish oil. .sup.1H NMR (400 MHz, CDCl.sub.3): =0.87 (t, J=6 Hz, 3H), 1.26-1.38 (m, 14H), 1.51-1.66 (m, 4H), 2.07-2.12 (m, 2H), 2.30 (t, J=7.5 Hz, 2H), 3.63 (t, J=6.6 Hz, 2H), 4.61-4.62 (m, 2H), 5.48-5.55 (m, 1H), 5.60-5.67 (m, 1H) ppm. .sup.13C NMR (101 MHz, CDCl.sub.3): =14.07, 22.60, 24.88, 25.54, 27.54, 28.86, 29.02, 29.07, 29.39, 31.68, 32.68, 34.30, 60.22, 62.97, 123.34, 135.45, 173.75 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3637 w, 3453 w ( OH); 1056 m ( COH); 1736 vs ( CO); 1238 m, 1170 s ( CO); 3025 w (.sub.as=CH); 1659 w ( CC); 1419 w (=CH); 2955 s (.sub.asCH.sub.3); 2931 vs (.sub.a CH.sub.2); 2858 s (.sub.s CH.sub.2); 2872 s, sh (.sub.s CH.sub.3); 1466 m and 1457 m (.sub.s CH.sub.2 and .sub.as CH.sub.3); 1378 m (.sub.s CH.sub.3); 722 m (.sub.as and .sub.as CH.sub.2). HRMS (EI): m/z calculated for C.sub.17H.sub.32O.sub.3 [M].sup.+ 284.2351; found 284.2355.

(Z)-Non-2-en-1-yl-8-oxoocktanoate 18

(46) Dess-Martin periodinane (2.46 g, 5.80 mmol, 1.3 eq.) was added to a solution of alcohol 17 (1.27 ml, 4.46 mmol) in DCM (100 ml), cooled to 0 C. in 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 (10 g Na.sub.2S2O.sub.3.Math.5H.sub.2O/50 ml H.sub.2O) and saturated aqueous sodium bicarbonate solution (50 ml), and stirred for 1 h at rt until the initially milky solution turned clear. The solution was poured into a 500 ml separatory flask, diluted with water (100 ml), and the product was extracted with DCM (100 ml, 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 in an RVE. The crude product was purified by silica gel column chromatography using a linear gradient of ethyl acetate in cyclohexane (0-30%). Aldehyde 18 (0.573 g, 45.4%; R.sub.f 0.49 in mobile phase CE20, detection with KMnO.sub.4) was obtained as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3): =0.88 (t, J=7 Hz, 3H), 1.26-1.38 (m, 14H), 1.59-1.67 (m, 4H), 2.07-2.12 (m, 2H), 2.30 (t, J=7.5 Hz, 2H), 2.40-2.44 (m, 2H), 4.61-4.63 (m, 2H), 5.48-5.55 (m, 1H), 5.61-5.68 (m, 1H), 9.76 (t, J=1.8 Hz, 1H) ppm. .sup.13C NMR (101 MHz, CDCl.sub.3): =14.07, 21.86, 22.60, 24.71, 27.54, 28.77, 28.83, 28.86, 29.39, 31.68, 34.19, 43.79, 60.25, 123.31, 135.49, 173.59, 202.62 ppm. IR (CCl.sub.4): .sub.m/cm.sup.1=2818 m, 2716 m (aldeh, CH); 1733 vs (aldeh, CO); 1395 m, sh (aldeh, OCH); 1733 vs ( CO); 1244 m, 1172 s ( CO); 3026 m (.sub.as=CH); 1659 w ( CC); 2956 s (.sub.as CH.sub.3); 2930 vs (.sub.as CH.sub.2); 2858 s (.sub.s CH.sub.2); 2873 s, sh (.sub.s CH.sub.3); 1466 m a 1462 m (.sub.s CH.sub.2 a .sub.as CH.sub.3); 1378 m a 1373 m (.sub.s CH.sub.3). HRMS (ESI): m/z calculated for C.sub.17H.sub.29O.sub.4 [MH].sup. 297.20713; found 297.20722.

N.SUP.1.,N.SUP.1.-Bis(8-((Z)-non-2-en-1-yl)oxy-8-oxooctyl)hexane-1,6-diamine 20

(47) Amine 19 was prepared from amine 1e (140 mg, 0.647 mmol), aldehyde 18 (0.548 g, 1.94 mmol, 3 eq.) and sodium triacetoxyborohydride (0.411 g, 1.94 mmol, 3 eq.) according to the procedure described for 2a in Example 1, except that the reaction mixture was evaporated without prior extraction and the residue was directly purified by chromatography. Amine 19 was obtained as a slightly yellowish oil (0.446 g, 92.0%).

(48) The deprotection of amine 19 was performed in a mixture of TFA (4 ml) and DCM (4 ml) according to the procedure described for 2a in Example 1; diamine 20 (0.333 g, 86.2%; R.sub.f 0.16 in mobile phase D2, detection with ninhydrin) was obtained as a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =5.63, 5.50, 4.61, 3.06, 3.02, 2.99, 2.29, 2.08, 1.82, 1.74, 1.60, 1.57, 1.42, 1.34, 1.28, 1.27, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =173.59, 135.39, 129.29, 60.24, 52.64, 52.19, 39.50, 34.12, 31.64, 29.34, 28.84, 28.82, 28.73, 27.51, 26.60, 25.76, 25.36, 24.69, 23.11, 22.57, 14.05 ppm. IR (CCl.sub.4): .sub.m/cm.sup.1=3446 w (.sub.as NH.sub.2); 1612 w (.sub.s NH.sub.2); 2800 m, sh (.sub.s NCH.sub.2); 1736 vs ( CO); 1236 m, 1168 s ( CO); 3024 m (.sub.as=CH); 1679 w ( CC); 1419 w (=CH); 2957 s (.sub.as CH.sub.3); 2931 vs (.sub.as CH.sub.2); 2858 s (.sub.s CH.sub.2); 2873 s, sh (.sub.s CH.sub.3); 1467 m and 1457 m (.sub.s CH.sub.2 and .sub.as CH.sub.3); 1378 m (.sub.s CH.sub.3); 721 w (.sub.as and .sub.as CH.sub.2). HRMS (ESI): m/z calculated for C.sub.40H.sub.77O.sub.4N.sub.2 [M+H].sup.+ 649.58779; found 649.58767.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(bis(8-((Z)-non-2-en-1-yl)oxy-8-oxooctyl)amino)hexyl)adamantane-1,3,5-tricarboxamide 21

(49) Lipidoid 21 was prepared from adamantane-1,3,5-tricarboxylic acid (30 mg, 0.112 mmol), HCTU (192 mg, 0.447 mmol, 4 eq.), DIPEA (0.312 ml, 1.79 mmol, 16 eq.) and diamine 20 (290 mg, 0.447 mmol, 4 eq.) according to the procedure described for compound 4a in Example 1; lipidoid 21 (181 mg, 74.9%; R.sub.f 0.29 in mobile phase D2, detection with ninhydrin) was obtained as a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =7.54, 5.63, 5.50, 4.61, 3.29, 3.00, 2.98, 2.34, 2.29, 2.23, 2.08, 1.98, 1.89, 1.76, 1.60, 1.585, 1.42, 1.38, 1.35, 1.28, 1.26, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =177.29, 173.53, 135.41, 123.27, 60.25, 52.62, 52.27, 41.72, 39.57, 39.20, 37.12, 34.09, 31.64, 29.34, 28.85, 28.82, 28.74, 28.44, 28.37, 27.51, 26.63, 25.83, 25.56, 24.67, 23.11, 22.57, 14.06 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3321 w ( NH); 1644 m (amide I); 1535 w-m (amide II); 1736 s ( CO); 1276 w-m, 1166 m ( CO); 3025 w (.sub.as=CH); 1419 w (=CH); 2956 s, sh (.sub.as CH.sub.3); 2930 vs (.sub.as CH.sub.2); 2858 s (.sub.s CH.sub.2); 2873 m, sh (.sub.sCH.sub.3); 1467 m and 1457 m (.sub.s CH.sub.2 and .sub.as CH.sub.3); 1378 w-m (.sub.s CH.sub.3). HRMS (MALDI): m/z calculated for C.sub.133H.sub.239N.sub.6O.sub.5 [M+H].sup.+ 2160.8118; found 2160.8164.

Example 11

3,5,7-Tris((6-(didodecylamino)hexyl)carbamoyl)adamantane-1-carboxylic acid 22

(50) Thionylchloride (300 l) and DMF (2 l) was added to adamantane-1,3,5,7-tetracarboxylic acid (21 mg, 0.067 mmol), and the suspension was stirred for 2 h at 70 C. in a closed vial; during this time the suspension turned into a clear homogeneous solution. 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 was dissolved in 0.5 ml of anhydrous DMF to form a clear solution. Then, a solution of N.sup.1,N.sup.1-didodecylhexane-1,6-diamine (76 mg, 0.168 mmol, 2.5 eq.) and DIPEA (117 l, 0.672 mmol, 10 eq.) in a mixture of DCM (1.5 ml) and DMF (0.5 ml), and the reaction mixture was stirred for 10 min at rt. The reaction mixture was then adsorbed onto silica (10 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 35-85%) to yield the target compound 22 (45 mg, 41.4%; R.sub.f 0.50 in D2/3, visualization by ninhydrin) as a thick pale yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =7.32, 3.24, 3.05-2.95, 2.16, 1.97, 1.92, 1.80-1.72, 1.55, 1.38-1.23, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.52, 52.89, 52.20, 42.28, 39.39, 38.96, 31.89, 29.60, 29.50, 29.44, 29.32, 29.10, 28.72, 26.83, 26.23, 25.99, 23.47, 23.14, 22.66, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3314 w, br ( NH), 1643 m (amide I) and 1638 w (amide II), 2954 s (.sub.as CH.sub.3), 2927 vs (.sub.asCH.sub.2), 2855 s (.sub.s CH.sub.2), 1467 m, sh and 1457 m, sh (.sub.s CH.sub.2 and .sub.as CH.sub.3), 1378 w (.sub.s CH.sub.3); COOH dimer: 2591 w, br ( OH), 1715 w ( CO), 1411 vw and 1283 w, br ( COH and CCO). HRMS (MALDI): m/z calcd. for C.sub.104H.sub.203N.sub.6O.sub.5 [M+H].sup.+ 1616.5810; found 1616.58014.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(didodecylamino)hexyl)adamantane-1,3,5,7-tetracarboxamide 23

(51) DMF (2 l) and thionylchloride (24 l, 0.349 mmol, 14 eq.) were added to a solution of acid 22 (27 mg, 0.017 mmol) in DCM (3.5 ml), and the suspension was stirred for 2 h at rt in a closed vial. The solution was then bubbled with a gentle stream of anhydrous NH.sub.3 for 5 min; a white precipitate formed immediately. After 10 min, the reaction mixture was filtered through a celite pad, the filtrate was adsorbed onto silica (10 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 30-70%) to yield target compound 23 (15 mg, 55.6%; R.sub.f 0.57 in D2/3, visualization by ninhydrin) as a thick pale yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =6.69, 6.49, 3.21, 2.67, 1.99, 1.96, 1.57, 1.51, 1.33, 1.285, 1.245, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =178.41, 175.71, 53.42, 53.07, 42.38, 39.52, 39.40, 39.18, 31.88, 29.62, 29.60, 29.58, 29.55, 29.37, 29.32, 29.02, 27.24, 26.56, 26.21, 22.66, 14.09 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3330 w, br ( NH), 1649 m (amide I) and 1536 w (amide II), 2953 m (.sub.as CH.sub.3), 2927 vs (.sub.asCH.sub.2), 2875 m, sh (.sub.s CH.sub.3), 2855 s and 2805 vw (.sub.s CH.sub.2), 1467 m, and 1457 w, 1438 w, sh (.sub.s CH.sub.2 and .sub.as CH.sub.3), 1378 w (.sub.s CH.sub.3); prim. amide: 3199 w, br ( NH.sub.2 viz.), 1697 vw, sh ( CO), 1605 w, sh (.sub.s NH.sub.2). HRMS (MALDI): m/z calcd. for C.sub.104H.sub.204N.sub.7O.sub.4 [M+H].sup.+ 1615.5969; found 1615.5998.

Example 12

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(didodecylamino)hexyl)-N.SUP.7.-(2-hydroxyethyl)adamantane-1,3,5,7-tetracarboxamide 24

(52) DMF (2 l) and thionylchloride (20 l, 0.286 mmol, 14 eq.) were added to a solution of acid 22 (33 mg, 0.020 mmol) in DCM (1.5 ml), and the suspension was stirred for 1 h at rt in a closed vial. Ethanolamine (50 l, 0.816 mmol, 40 eq.) was then added, and a white precipitate formed immediately. After 20 min, the reaction mixture was filtered through a celite pad, the filtrate was adsorbed onto silica (10 g), and the solvents were evaporated in vacuo. The crude product was purified by flash chromatography on silica (elution with a linear gradient of D1 in DCM, 35-85%) to yield the target compound 24 (31 mg, 91.5%; R.sub.f 0.59 in D2/3, visualization by ninhydrin) as a thick pale yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =7.39, 6.92, 3.69, 3.37, 3.22, 3.00, 2.02, 1.96, 1.76, 1.53, 1.40-1.22, 0.86 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.76, 176.16, 61.73, 61.38, 52.73, 52.16, 43.13, 42.55, 42.41, 39.54, 39.43, 38.74, 31.86, 29.56, 29.45, 29.40, 29.28, 29.06, 28.64, 26.78, 25.92, 25.64, 23.38, 23.08, 22.64, 14.07 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3324 w, br ( NH), 1646 m (amide I) and 1538 w (amide II), 2955 s (.sub.asCH.sub.3), 2927 vs (.sub.as CH.sub.2), 2855 s and 2876 m, sh (.sub.s CH.sub.3), 1467 m, 1457 m and 1435 w, sh (.sub.s CH.sub.2 and .sub.as CH.sub.3), 1378 w (.sub.s CH.sub.3), 1060 w, br, 1036 w, br ( COH). HRMS (MALDI): m/z calcd. for C.sub.106H.sub.208N.sub.7O.sub.5 [M+H].sup.+ 1659.6231; found 1659.62718.

Example 13

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(didodecylamino)hexyl)-N.SUP.7.-(1,3-dihydroxypropan-2-yl)adamantane-1,3,5,7-tetracarboxamide 25

(53) Following the procedure outlined for 24, the target compound 25 was prepared from acid 22 (51 mg, 0.031 mmol), and serinol (115 mg, 1.26 mmol, 40 eq.) to yield lipidoid 25 as a thick pale yellow oil (19 mg, 35.6%; R.sub.f 0.59 in D2/3). .sup.1H NMR (600 MHz, CDCl.sub.3): =7.79, 7.21, 7.17, 7.08, 3.26, 3.01, 2.03, 1.92, 1.775, 1.57, 1.385, 1.33, 1.28-1.24 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.14, 175.04, 52.74, 52.66, 52.32, 52.17, 39.28-38.65, 31.89, 29.68, 29.59, 29.49, 29.43, 29.32, 29.09, 28.47, 26.83, 26.80, 25.98-25.40, 23.05, 22.67, 14.10 ppm. IR (CCl.sub.4): .sub.max/cm.sup.1=3324 w, br ( NH), 1646 m (amide I) and 1538 w (amide II), 2955 s (.sub.as CH.sub.3), 2927 vs (.sub.as CH.sub.2), 2855 s and 2876 m, sh (.sub.s CH.sub.3), 1467 m, 1457 m and 1435 w, sh (.sub.s CH.sub.2 and .sub.as CH.sub.3), 1378 w (.sub.s CH.sub.3), 1060 w, br, 1036 w, br ( COH). HRMS (MALDI): m/z calcd. for C.sub.107H.sub.210N.sub.7O.sub.6 [M+H].sup.+ 1689.6337; found 1616.63235.

Example 14

N.SUP.1.,N.SUP.1.-Di((heptyloxycarbonyl)propyl)hexane-1,6-diamine 28a

(54) Diisopropylcarbodiimide (3.17 ml, 20.24 mmol, 1.3 eq.) and DMAP (57.1 mg, 0.467 mmol, 0.03 eq.) were added to a solution of 4-bromobutyric acid (2.60 g, 15.57 mmol) and 1-heptanol (2.64 ml, 18.68 mmol, 1.2 eq.) in DCM (60 ml), and the mixture was stirred at rt for 1 h. 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 ethylacetate in cyclohexane, 0-10%) to yield the target compound 26a (3.916 g, 94.8%; R.sub.f 0.36 in CE5, visualization by KMnO.sub.4) as a colorless oil.

(55) Bromoester 26a (1.53 g, 5.78 mmol, 2.5 eq.) and potassium carbonate (3.19 g, 23.11 mmol, 10 eq.) were added to a solution of N-Boc-1,6-diaminohexane (0.50 g, 2.31 mmol) in ACN (10 ml), and the mixture was stirred at 35 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 (40 g, elution with a linear gradient of ethylacetate in cyclohexane, 0-100%) to yield the target compound 27a (1.093 g, 80.9%; R.sub.f 0.35 in in CE50 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin) as a pale yellow oil.

(56) The deprotection of amine 27a was performed according to the procedure described for compound 2a in Example 1; diamine 28a (0.817 g, 90.2%; R.sub.f 0.27 in mobile phase D2, detection with ninhydrin) was obtained in the form of a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =4.05, 3.63, 3.21, 3.15, 2.91, 2.85, 2.67, 2.53-2.41, 2.32, 1.86, 1.76, 1.61, 1.46, 1.35-1.25, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =173.66, 64.58, 53.66, 52.98, 52.87, 40.78, 40.34, 31.87, 31.70, 30.10, 28.90, 28.62, 26.85, 26.45, 25.87, 22.56, 22.02, 14.04 ppm. HRMS (ESI): m/z calcd. for C.sub.28H.sub.57N.sub.2O.sub.4 [M+H].sup.+ 485.43128; found 485.43039.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(bis((heptyloxycarbonyl)propyl)amino)hexyl)adamantane-1,3,5-tricarboxamide 29a

(57) Thionylchloride (300 l) and DMF (2 l) was added to adamantane-1,3,5-tricarboxylic acid (60 mg, 0.224 mmol), and the suspension was stirred for 1 h at 70 C. in a closed vial; during this time the suspension turned into a clear homogeneous solution. 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 was dissolved in 0.5 ml of anhydrous DMF to form a clear solution. Then, a solution of amine 28a (434 mg, 0.895 mmol, 4 eq.) and DIPEA (390 l, 2.24 mmol, 10 eq.) in a mixture of DCM (1.5 ml) and DMF (0.5 ml), and the reaction mixture was stirred for 10 min 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 (40 g, elution with a linear gradient of D1 in DCM, 20-60%) to yield the target compound 29a (115 mg, 30.8%; R.sub.f 0.51 in D2, visualization by ninhydrin) as a thick pale yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =5.76, 4.05, 3.21, 2.42, 2.32, 2.01, 1.84, 1.80, 1.74, 1.61, 1.47, 1.41, 1.34-1.25, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.01, 173.73, 64.53, 53.72, 53.00, 41.65, 39.77, 39.48, 37.88, 31.96, 31.70, 29.51, 28.90, 28.63, 27.02, 26.76, 25.87, 22.56, 14.05 ppm. HRMS (MALDI): m/z calcd. for C.sub.97H.sub.179N.sub.6O.sub.15 [M+H].sup.+ 1668.3423; found 1668.3398.

Example 15

N.SUP.1.,N.SUP.1.-Di((hexyloxycarbonyl)butyl)hexane-1,6-diamine 28b

(58) Following the procedure outlined for 26a, bromoester 26b was prepared from 5-bromopentanoic acid (3.0 g, 16.57 mmol), 1-hexanol (2.48 ml, 19.89 mmol, 1.2 eq.), DIC (3.37 ml, 21.54 mmol, 1.3 eq.) and DMAP (61 mg, 0.497 mmol, 0.03 eq.) to yield 26b as a colorless oil (3.938 g, 89.6%, R.sub.f 0.28 in CE5, visualization by KMnO.sub.4).

(59) Following the procedure outlined for 27a, Boc-derivative 27b was prepared from bromoester 26b (1.53 g, 5.78 mmol, 2.5 eq.), N-Boc-1,6-diaminohexane (0.50 g, 2.31 mmol) and potassium carbonate (3.19 g, 23.11 mmol, 10 eq.) to yield 27b as a pale yellow oil (1.080 g, 79.9%, R.sub.f 0.30 in CE50 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin).

(60) The deprotection of amine 27b was performed according to the procedure described for compound 2a in Example 1; diamine 28b (0.788 g, 86.9%; R.sub.f 0.13 in mobile phase D2, detection with ninhydrin) was obtained in the form of 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. HRMS (ESI): m/z calcd. for C.sub.28H.sub.57N.sub.2O.sub.4 [M+H].sup.+ 485.43128; found 485.43052.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(bis((hexyloxycarbonyl)butyl)amino)hexyl)adamantane-1,3,5-tricarboxamide 29b

(61) Following the procedure outlined for 29a in Example 14, lipidoid 29b was prepared from adamantane-1,3,5-tricarboxylic acid (53 mg, 0.198 mmol), amine 28b (383 mg, 0.790 mmol, 4 eq.) and DIPEA (344 l, 1.98 mmol, 10 eq.) to yield 29b as a thick pale yellow oil (191 mg, 57.9%, R.sub.f 0.48 in D2, visualization by ninhydrin). .sup.1H NMR (600 MHz, CDCl.sub.3): =5.72, 4.05, 3.21, 2.38, 2.35, 2.30, 2.01, 1.83, 1.80, 1.61, 1.44, 1.40, 1.35-1.27, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =175.96, 173.77, 64.44, 53.98, 53.63, 41.64, 39.73, 39.55, 37.92, 34.27, 31.42, 29.59, 28.60, 27.22, 26.89, 26.63, 25.58, 23.01, 22.52, 13.99 ppm. HRMS (MALDI): m/z calcd. for C.sub.97H.sub.179N.sub.6O.sub.15 [M+H].sup.+ 1668.3428; found 1668.3418.

Example 16

N.SUP.1.,N.SUP.1.-Di((nonyloxycarbonyl)propyl)hexane-1,6-diamine 28c

(62) Following the procedure outlined for 26a, bromoester 26c was prepared from 4-bromobutyric acid (2.60 g, 15.57 mmol), 1-nonanol (3.26 ml, 18.68 mmol, 1.2 eq.), DIC (3.17 ml, 20.24 mmol, 1.3 eq.) and DMAP (57 mg, 0.467 mmol, 0.03 eq.) to yield 26c as a colorless oil (4.025 g, 88.2%, R.sub.f 0.32 in CE5, visualization by KMnO.sub.4).

(63) Following the procedure outlined for 27a, Boc-derivative 27c was prepared from bromoester 26c (1.25 g, 4.28 mmol, 2.5 eq.), N-Boc-1,6-diaminohexane (0.370 g, 1.71 mmol) and potassium carbonate (2.36 g, 17.10 mmol, 10 eq.) to yield 27c as a pale yellow oil (0.848 g, 77.3%, R.sub.f 0.45 in CE50 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin).

(64) The deprotection of amine 27c was performed according to the procedure described for compound 2a in Example 1; diamine 28c (0.631 g, 88.2%; R.sub.f 0.29 in mobile phase D2, detection with ninhydrin) was obtained in the form of a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =4.66, 4.05, 3.16, 2.91, 2.63, 2.59, 2.53, 2.48, 2.34, 1.81, 1.70, 1.61, 1.54, 1.47, 1.42, 1.35-1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =173.39, 64.72, 64.66, 52.53, 31.84, 31.59, 29.46, 29.25, 29.22, 28.61, 28.60, 25.91, 25.90, 22.64, 14.08 ppm. HRMS (ESI): m/z calcd. for C.sub.32H.sub.65N.sub.2O.sub.4 [M+H].sup.+ 541.49388; found 541.49303.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(bis((nonyloxycarbonyl)propyl)amino)hexyl)adamantane-1,3,5-tricarboxamide 29c

(65) Following the procedure outlined for 29a in Example 14, lipidoid 29c was prepared from adamantane-1,3,5-tricarboxylic acid (42 mg, 0.157 mmol), amine 28c (339 mg, 0.626 mmol, 4 eq.) and DIPEA (273 l, 1.57 mmol, 10 eq.) to yield 29c as a thick pale yellow oil (134 mg, 46.6%, R.sub.f 0.56 in D2, visualization by ninhydrin). .sup.1H NMR (600 MHz, CDCl.sub.3): =5.72, 4.05, 3.21, 2.40, 2.36, 2.31, 2.01, 1.83, 1.80, 1.72, 1.61, 1.47, 1.38, 1.33-1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =175.96, 173.84, 64.48, 53.80, 53.12, 41.65, 39.74, 39.54, 37.92, 32.06, 31.84, 29.47, 29.26, 29.22, 28.64, 25.92, 22.64, 14.09 ppm. HRMS (MALDI): m/z calcd. for C.sub.109H.sub.203N.sub.6O.sub.15 [M+H].sup.+ 1836.5306; found 836.5319.

Example 17

N.SUP.1.,N.SUP.1.-Di((octyloxycarbonyl)butyl)hexane-1,6-diamine 28d

(66) Following the procedure outlined for 26a, bromoester 26d was prepared from 5-bromopentanoic acid (3.0 g, 16.57 mmol), 1-octanol (3.13 ml, 19.89 mmol, 1.2 eq.), DIC (3.37 ml, 21.54 mmol, 1.3 eq.) and DMAP (61 mg, 0.497 mmol, 0.03 eq.) to yield 26d as a colorless oil (4.327 g, 89.0%, R.sub.f 0.32 in CE5, visualization by KMnO.sub.4).

(67) Following the procedure outlined for 27a, Boc-derivative 27d was prepared from bromoester 26d (1.73 g, 5.89 mmol, 2.5 eq.), N-Boc-1,6-diaminohexane (0.510 g, 2.36 mmol) and potassium carbonate (3.26 g, 23.58 mmol, 10 eq.) to yield 27d as a pale yellow oil (1.241 g, 82.1%, R.sub.f 0.18 in CE50 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin).

(68) The deprotection of amine 27d was performed according to the procedure described for compound 2a in Example 1; diamine 28d (1.09 g, quantitative; R.sub.f 0.22 in mobile phase D2, detection with ninhydrin) was obtained in the form of a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =8.22, 4.86, 4.05, 3.04, 2.37, 1.75, 1.67, 1.61, 1.48, 1.40, 1.32-1.25, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =172.99, 64.85, 52.44, 51.90, 39.41, 33.13, 31.75, 29.18, 29.14, 28.54, 26.54, 25.86, 25.49, 24.88, 22.60, 21.83, 14.05 ppm. HRMS (ESI): m/z calcd. for C.sub.32H.sub.65N.sub.2O.sub.4 [M+H].sup.+ 541.49388; found 541.49297.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(bis((octyloxycarbonyl)butyl)amino)hexyl)adamantane-1,3,5-tricarboxamide 29d

(69) Following the procedure outlined for 29a in Example 14, lipidoid 29d was prepared from adamantane-1,3,5-tricarboxylic acid (70 mg, 0.261 mmol), amine 28d (565 mg, 1.04 mmol, 4 eq.) and DIPEA (455 l, 2.61 mmol, 10 eq.) to yield 29d as a thick pale yellow oil (229 mg, 47.8%, R.sub.f 0.58 in D2, visualization by ninhydrin). .sup.1H NMR (600 MHz, CDCl.sub.3): =5.79, 4.05, 3.21, 2.44, 2.31, 2.01, 1.84, 1.80, 1.61, 1.47, 1.34-1.24, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.03, 173.63, 64.50, 53.54, 41.65, 39.77, 39.45, 37.88, 34.12, 31.76, 29.48, 29.20, 29.16, 28.63, 25.91, 22.87, 22.61, 14.07 ppm. HRMS (ESI): m/z calcd. for C.sub.109H.sub.203N.sub.6O.sub.15 [M+H].sup.+ 1836.53010; found 1836.52959.

Example 18

N.SUP.1.,N.SUP.1.-Di((heptyloxycarbonyl)pentyl)hexane-1,6-diamine 28e

(70) Following the procedure outlined for 26a, bromoester 26e was prepared from 6-bromohexanoic acid (3.0 g, 15.38 mmol), 1-heptanol (2.61 ml, 18.46 mmol, 1.2 eq.), DIC (3.13 ml, 19.99 mmol, 1.3 eq.) and DMAP (56 mg, 0.461 mmol, 0.03 eq.) to yield 26e as a colorless oil (4.177 g, 92.6%, R.sub.f 0.27 in CE5, visualization by KMnO.sub.4).

(71) Following the procedure outlined for 27a, Boc-derivative 27e was prepared from bromoester 26e (1.49 g, 5.08 mmol, 2.5 eq.), N-Boc-1,6-diaminohexane (0.440 g, 2.03 mmol) and potassium carbonate (2.81 g, 20.34 mmol, 10 eq.) to yield 27e as a pale yellow oil (1.076 g, 82.5%, R.sub.f 0.15 in CE50 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin).

(72) The deprotection of amine 27e was performed according to the procedure described for compound 2a in Example 1; diamine 28e (0.895 g, 98.6%; R.sub.f 0.17 in mobile phase D2, detection with ninhydrin) was obtained in the form of a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =8.40, 4.04, 3.04, 2.99, 2.31, 2.05, 1.72, 1.66, 1.61, 1.48, 1.39, 1.33-1.26, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =173.38, 64.64, 52.02, 33.78, 31.68, 28.88, 28.58, 26.70, 26.25, 26.13, 25.83, 25.68, 24.93, 24.20, 22.99, 22.55, 22.48, 14.03 ppm. HRMS (ESI): m/z calcd. for C.sub.32H.sub.65N.sub.2O.sub.4 [M+H].sup.+ 541.49388; found 541.49303.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(bis((heptyloxycarbonyl)pentyl)amino)hexyl)adamantane-1,3,5-tricarboxamide 29e

(73) Following the procedure outlined for 29a in Example 14, lipidoid 29e was prepared from adamantane-1,3,5-tricarboxylic acid (55 mg, 0.205 mmol), amine 28e (444 mg, 0.820 mmol, 4 eq.) and DIPEA (357 l, 2.05 mmol, 10 eq.) to yield 29e as a thick pale yellow oil (150 mg, 39.8%, R.sub.f 0.37 in D2, visualization by ninhydrin). .sup.1H NMR (600 MHz, CDCl.sub.3): =5.72, 4.05, 3.21, 2.37, 2.29, 2.01, 1.83, 1.80, 1.62, 1.47, 1.42, 1.33-1.25, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =175.97, 173.84, 64.42, 53.96, 41.64, 39.74, 39.53, 37.91, 34.35, 31.70, 29.59, 28.89, 28.63, 27.14, 25.87, 24.97, 22.56, 14.05 ppm. HRMS (ESI): m/z calcd. for C.sub.109H.sub.203N.sub.6O.sub.15 [M+H].sup.+ 1836.53010; found 1836.52930.

Example 19

N.SUP.1.,N.SUP.1.-Di(((nonan-3-yl)oxycarbonyl)propyl)hexane-1,6-diamine 28f

(74) Following the procedure outlined for 26a, bromoester 26f was prepared from 6-bromohexanoic acid (3.2 g, 16.41 mmol), 3-nonanol (2.60 g, 18.05 mmol, 1.1 eq.), DIC (3.27 ml, 21.33 mmol, 1.3 eq.) and DMAP (60 mg, 0.492 mmol, 0.03 eq.) to yield 26f as a colorless oil (2.88 g, 54.5%, R.sub.f 0.42 in CE5, visualization by KMnO.sub.4).

(75) Following the procedure outlined for 27a, Boc-derivative 27f was prepared from bromoester 26f (2.82 g, 8.78 mmol, 2.5 eq.), N-Boc-1,6-diaminohexane (0.760 g, 3.51 mmol) and potassium carbonate (4.86 g, 35.13 mmol, 10 eq.) to yield 27f as a pale yellow oil (1.72 g, 70.19%, R.sub.f 0.56 in CE50 on an NH.sub.3-pretreated TLC plate, visualization by ninhydrin).

(76) The deprotection of amine 27f was performed according to the procedure described for compound 2a in Example 1; diamine 28f (1.84 g, quantitative; R.sub.f 0.25 in mobile phase D2, detection with ninhydrin) was obtained in the form of a yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =8.29, 4.80, 2.99, 2.305, 1.69-1.66, 1.55-1.51, 1.445, 1.38, 1.28-1.24, 0.87, 0.86 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =173.14, 75.61, 52.10, 39.27, 34.08, 33.56, 31.71, 29.15, 26.90, 26.74, 26.14, 25.69, 25.27, 24.86, 24.33, 22.97, 22.56, 22.51, 14.03, 9.57 ppm. HRMS (ESI): m/z calcd. for C.sub.36H.sub.73N.sub.2O.sub.4 [M+H].sup.+ 597.55649; found 597.55631.

N.SUP.1.,N.SUP.3.,N.SUP.5.-Tris(6-(bis(((nonan-3yl)oxycarbonyl)propyl)amino)hexyl)adamantane-1,3,5-tricarboxamide 29f

(77) Following the procedure outlined for 29a in Example 14, lipidoid 29f was prepared from adamantane-1,3,5-tricarboxylic acid (50 mg, 0.186 mmol), amine 28f (334 mg, 746 mmol, 4 eq.) and DIPEA (325 l, 1.86 mmol, 10 eq.) to yield 29f as a thick pale yellow oil (237 mg, 63.4%, R.sub.f 0.44 in D2, visualization by ninhydrin). .sup.1H NMR (600 MHz, CDCl.sub.3): =6.385, 4.79, 3.21, 2.99, 2.305, 2.015, 1.88, 1.80, 1.73, 1.66, 1.55-1.50, 1.38-1.35, 1.27-1.24, 0.865, 0.855 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.57, 173.02, 76.58, 52.43, 51.98, 41.69, 39.89, 38.89, 37.64, 34.09, 33.55, 31.70, 29.14, 28.84, 26.90, 26.28, 26.01, 25.78, 24.39, 23.13, 22.96, 22.55, 14.04, 9.59 ppm. HRMS (MALDI): m/z calcd. for C.sub.121H.sub.227N.sub.6O.sub.15 [M+H].sup.+ 2004.7179; found 2004.7187.

Example 20

6-(Di((heptyloxycarbonyl)propyl)amino)hexan-1-ol 30a

(78) Bromoester 26a (1.13 g, 4.27 mmol, 2.5 eq.) and potassium carbonate (2.36 g, 17.07 mmol, 10 eq.) were added to a solution of 6-aminohexan-1-ol (0.20 g, 1.71 mmol) in ACN (10 ml), and the mixture was stirred at 50 C. for 20 h. 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 (40 g, elution with a linear gradient of D1 in DCM, 0-50%) to yield the target compound 30a (0.552 g, 63.0%; R.sub.f 0.56 in D2, visualization by KMnO.sub.4) as a pale yellow oil.

(79) .sup.1H NMR (600 MHz, CDCl.sub.3): =4.05, 3.63, 2.52, 2.33, 1.80, 1.61, 1.565, 1.50, 1.375, 1.32-1.27, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =173.54, 64.63, 53.45, 52.82, 32.59, 31.78, 31.69, 28.89, 28.60, 26.95, 25.86, 25.45, 22.55, 21.78, 14.03 ppm. HRMS (ESI): m/z calcd. for C.sub.2H.sub.56NO.sub.5 [M+H].sup.+ 486.41530; found 486.41467.

tris(6-(Di((heptyloxycarbonyl)propyl))hexyl)adamantane-1,3,5-tricarboxylate 31a

(80) Tetramethylfluoroformamidinium hexafluorophosphate (TFFH, 169 mg, 0.640 mmol, 3.3 eq.) and DIPEA (0.506 ml, 2.91 mmol, 15 eq.) were added to a solution of adamantane-1,3,5-tricarboxylic acid (52 mg, 0.194 mmol) in anhydrous DMF (5 ml), and the solution was stirred for 30 min at 0 C. Then a solution of alcohol 30a (311 mg, 0.640 mmol, 3.3 eq.) and DMAP (7 mg, 0.058 mmol, 0.3 eq.) in DMF (2.0 ml) was added, and the reaction mixture was stirred for 12 h at rt. The solution was poured into a 250 ml separatory flask, diluted with saturated aqueous NaHCO.sub.3 (50 ml), and the product was extracted with diethyl ether (100 ml, 250 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 crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (0-65%). Lipidoid 31a (33 mg, 10.3%; R.sub.f 0.57 in mobile phase D4, detection with ninhydrin) was obtained in the form of a viscous yellowish oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =4.05, 2.40, 2.05-1.94, 1.83, 1.61, 1.35-1.25, 0.886 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.02, 64.95, 64.45, 41.35, 39.14, 37.14, 31.67, 28.87, 28.55, 25.83, 22.54, 14.03 ppm. HRMS (MALDI): m/z calcd. for C.sub.97H.sub.176N.sub.3O.sub.18 [M+H].sup.+ 1671.2943; found 1671.2973.

Example 21

6-(Di((hexyloxycarbonyl)butyl)amino)hexan-1-ol 30b

(81) Following the procedure outlined for 30a, alcohol 30b was prepared from 6-aminohexan-1-ol (0.20 g, 1.71 mmol), bromoester 26b (1.13 g, 4.27 mmol, 2.5 eq.) and K.sub.2CO.sub.3 (2.36 g, 17.07 mmol, 10 eq.) to yield 30b as a pale yellow oil (0.599 g, 72.3%, R.sub.f 0.55 in D2, visualization by KMnO.sub.4). .sup.1H NMR (600 MHz, CDCl.sub.3): =4.04, 3.63, 2.46, 2.32, 1.76, 1.60, 1.56, 1.45, 1.37-1.25, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =173.69, 64.56, 62.79, 53.57, 52.96, 32.64, 31.90, 31.82, 29.45, 29.24, 29.21, 28.61, 27.02, 25.91, 25.51, 22.63, 14.07 ppm.

Tris(6-(di((hexyloxycarbonyl)butyl))hexyl)adamantane-1,3,5-tricarboxylate 31b

(82) Lipidoid 31b was prepared from adamantane-1,3,5-tricarboxylic acid (84 mg, 0.313 mmol), TFFH (273 mg, 1.03 mmol, 3.3 eq.), DIPEA (0.818 ml, 4.179 mmol, 15 eq.), DMAP (11 mg, 0.093 mmol, 0.3 eq.) and alcohol 30b (608 mg, 1.25 mmol, 4 eq.) according to the procedure described for compound 31a in Example 20; lipidoid 31b (55 mg, 10.4%; R.sub.f 0.74 in mobile phase D2, detection with ninhydrin) was obtained as a viscous yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =4.05, 2.96, 2.37, 2.33, 2.01, 1.94, 1.85-1.78, 1.66, 1.60, 1.38, 1.34-1.28, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.01, 172.90, 64.76-64.34, 52.16, 47.81, 46.61, 41.35, 39.14, 37.12, 33.22, 31.38, 28.53, 25.54, 22.50, 22.09, 13.97 ppm. HRMS (MALDI): m/z calcd. for C.sub.97H.sub.176N.sub.3O.sub.18 [M+H].sup.+ 1671.2943; found 1671.2910.

Example 22

6-(Di((nonyloxycarbonyl)propyl)amino)hexan-1-ol 30c

(83) Following the procedure outlined for 30a, alcohol 30c was prepared from 6-aminohexan-1-ol (0.20 g, 1.71 mmol), bromoester 26c (1.25 g, 4.27 mmol, 2.5 eq.) and K.sub.2CO.sub.3 (2.36 g, 17.07 mmol, 10 eq.) to yield 30c as a pale yellow oil (0.651 g, 70.4%, R.sub.f 0.59 in D2, visualization by KMnO.sub.4).

(84) .sup.1H NMR (600 MHz, CDCl.sub.3): =4.07, 3.65, 3.09, 3.03, 2.45, 2.16, 1.89, 1.61, 1.56, 1.45, 1.34-1.25, 0.88 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =172.24, 65.25, 62.39, 52.41, 51.55, 32.11, 31.84, 30.65, 29.45, 29.22, 28.54, 26.29, 25.87, 22.65, 18.39, 14.09 ppm.

Tris(6-(di((nonyloxycarbonyl)propyl))hexyl)adamantane-1,3,5-tricarboxylate 31c

(85) Lipidoid 31c was prepared from adamantane-1,3,5-tricarboxylic acid (86 mg, 0.320 mmol), TFFH (279 mg, 1.06 mmol, 3.3 eq.), DIPEA (0.838 ml, 4.81 mmol, 15 eq.), DMAP (12 mg, 0.096 mmol, 0.3 eq.) and alcohol 30c (695 mg, 1.28 mmol, 4 eq.) according to the procedure described for compound 31a in Example 20; lipidoid 31c (23 mg, 3.9%; R.sub.f 0.91 in mobile phase D2, detection with ninhydrin) was obtained as a viscous yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =4.05, 2.37, 2.01, 1.95, 1.83, 1.61, 1.35-1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.02, 64.70, 41.35, 39.15, 37.15, 31.82, 29.44, 29.23, 29.20, 28.58, 28.47, 25.88, 22.63, 14.07 ppm. HRMS (MALDI): m/z calcd. for C.sub.109H.sub.200N.sub.3O.sub.18 [M].sup.+ 1839.4821; found 1839.4799.

Example 23

6-(Di((octyloxycarbonyl)butyl)amino)hexan-1-ol 30d

(86) Following the procedure outlined for 30a, alcohol 30d was prepared from 6-aminohexan-1-ol (0.20 g, 1.71 mmol), bromoester 26d (1.25 g, 4.27 mmol, 2.5 eq.) and K.sub.2CO.sub.3 (2.36 g, 17.07 mmol, 10 eq.) to yield 30d as a pale yellow oil (0.623 g, 67.4%, R.sub.f 0.50 in D2, visualization by KMnO.sub.4).

(87) .sup.1H NMR (600 MHz, CDCl.sub.3): =4.05, 3.63, 2.47, 2.31, 1.61, 1.56, 1.50, 1.37-1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =173.65, 64.52, 62.78, 53.69, 53.42, 34.07, 32.60, 31.75, 29.19, 29.15, 28.61, 27.05, 25.90, 25.49, 22.85, 22.61, 14.06 ppm.

Tris(6-(di((octyloxycarbonyl)butyl))hexyl)adamantane-1,3,5-tricarboxylate 31d

(88) Lipidoid 31d was prepared from adamantane-1,3,5-tricarboxylic acid (83 mg, 0.309 mmol), TFFH (270 mg, 1.02 mmol, 3.3 eq.), DIPEA (0.808 ml, 4.64 mmol, 15 eq.), DMAP (11 mg, 0.093 mmol, 0.3 eq.) and alcohol 30d (671 mg, 1.24 mmol, 4 eq.) according to the procedure described for compound 31a in Example 20; lipidoid 31d (147 mg, 25.8%; R.sub.f 0.82 in mobile phase D2, detection with ninhydrin) was obtained as a viscous yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): =4.05, 2.34, 2.01, 1.95, 1.83, 1.78-1.56, 1.36-1.24, 0.87 ppm. .sup.13C NMR (150.9 MHz, CDCl.sub.3): =176.04, 64.76-64.46, 41.36, 39.16, 37.15, 31.75, 29.19, 29.15, 28.60, 25.90, 22.61, 14.06 ppm. HRMS (MALDI): m/z calcd. for C.sub.109H.sub.200N.sub.3O.sub.1s [M].sup.+ 1839.4821; found 1839.4792.

Example 24

(89) Preparation of Transfection Reagents

(90) Reagents were generated by mixing the individual components listed in Table 1 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 were used for the preparation. Only the DOPE-Cy5 stock solution had a concentration of 0.79 mM and was prepared in chloroform.

(91) TABLE-US-00001 TABLE 1 Composition of transfection reagents A01-A10. Concentration of individual components in transfection reagents (mM) compound A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 4a 1.1 4b 1.1 4c 1.1 4d 1.1 4e 1.1 4f 1.1 4g 1.1 9 1.1 13 1.1 21 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 DOPE-Cy5 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3

(92) TABLE-US-00002 TABLE 2 Composition of transfection reagents A11-A13. Concentration of individual components in transfection reagents (mM) compound A11 A12 A13 23 1.1 24 1.1 25 1.1 cholesterol 2.18 2.18 2.18 DOPE 1.65 1.65 1.65 DMG-PEG.sub.2000 0.075 0.075 0.075

(93) TABLE-US-00003 TABLE 3 Composition of transfection reagents A14-A23. Concentration of individual components in transfection reagents (mM) compound A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 29a 1.1 29b 1.1 29c 1.1 29d 1.1 29e 1.1 29f 1.1 31a 1.1 31b 1.1 31c 1.1 31d 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 DOPE-Cy5 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3 10.sup.3

(94) TABLE-US-00004 TABLE 4 Composition of transfection reagents A24-A29 (A26 and A27 are comparative examples). Concentration of individual components in transfection reagents (mM) compound A24 A25 A26 A27 A28 A29 4e 1.1 1.1 1.1 4d 1.1 D-Lin-MC3- 2.5 DMA TT3 1.1 cholesterol 2.18 2.18 1.93 2.18 2.18 2.18 DOPE 1.64 1.65 1.65 DOPC 1.64 DSPC 1.64 0.49 DMG-PEG.sub.2000 0.075 0.075 0.075 0.075 0.075 0.075 DOPE-Cy5 1.19 1.19 1.19 1.19 10.sup.3 10.sup.3 10.sup.3 10.sup.3

Example 25

(95) Preparation of Lipid Nanoparticles (LNP) Containing mRNA

(96) DNA encoding the fluorescent protein mKate2 was amplified from the plasmid pmKate2-C (Evrogen) using the primers (5-CGCCACCATGGTGAGCGAGCTG-3 (SEQ ID NO. 1); 5-CCTCCTCCACCTCTGTGCCCCAG-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.

(97) The mRNA-containing LNPs (mRNA-LNPs) were prepared as follows: 300 l of a solution of each of the A01-A27 transfection reagents prepared in Example 24 was mixed with a solution of 120 g of mRNA in 300 l of 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 mRNA solution were injected separately into each inlet by a linear pump at a constant flow rate of 300 l/min. The resulting 600 l nanoparticle solution was collected and immediately diluted by the addition of 600 l PBS; the corresponding nanoparticle samples designated B01-B27 were thus formed from the transfection reagents A01-A27. Each of the mRNA-LNP samples (B01-B27) was prepared in triplicate. The hydrodynamic diameter of freshly formed mRNA-LNPs was measured using dynamic light scattering (NanoZS Zetasizer, Malvern, Worcestershire, UK) at a scattering angle of 173 at 25 C. The hydrodynamic diameter of mRNA-LNPs ranged from 72 to 135 nm with the exception of B18 with diameter of 265 nm (Table 5). In this form, the particles were used for subsequent biological tests.

(98) TABLE-US-00005 TABLE 5 Hydrodynamic diameter of mRNA-LNPs including standard deviation measured by dynamic light scattering. LNP Diameter (nm) B01 81.5 8.0 B02 99.3 2.7 B03 85.1 4.1 B04 107.7 4.2 B05 76.1 1.9 B06 72.9 8.6 B07 73.6 3.9 B08 108.0 18.3 B09 90.8 5.5 B10 89.5 5.0 B11 82.5 0.2 B12 85.5 1.1 B13 102.1 10.7 B14 92.6 5.1 B15 118.0 5.5 B16 79.4 2.7 B17 72.2 4.0 B18 265.7 19.9 B19 71.8 4.2 B20 85.7 6.4 B21 135.1 1.1 B22 80.3 6.7 B23 80.1 5.4

Example 26

(99) Comparison of mRNA Transfection Using New LNPs In Vitro Using Various Helper Lipids in a Lipid Mixture

(100) LNPs B05, B24 and B25 containing mRNA encoding the fluorescent protein mKate2 prepared in triplicate in Example 25 were tested on cells of the human cell line HEK293. Cells were cultured in 96-well plates (510.sup.4 cells in 100 l culture medium per well) in IMDM medium supplemented with 10% FBS at 37 C. in 50% CO.sub.2. Cells were transfected with 2 l of mRNA-LNP (with a final total concentration of all lipid components of 20 M) and subsequently incubated for 24 hours. Transfections were performed in triplicates. The intensity of Cy5 fluorescence, indicating LNP entry into cells, and mKate2 fluorescence, indicating the translation of mRNA released from LNP after cell transfection, were analysed in a BD LSR Fortessa cytometer.

(101) The novel mRNA-LNPs, whose lipid mixture contained DOPE, DOPC or DSPC helper lipid, were able to efficiently transfect mRNA into the HEK293 cell line, with the most efficient transfection being achieved with mRNA-LNP containing DOPE helper lipid (Table 6).

(102) TABLE-US-00006 TABLE 6 Transfection efficiency of new mRNA-LNPs containing helper lipids DOPE (B05), DOPC (B24) or DSPC (B25). The indicated values of the fluorescence intensity of Cy5 and mKate2 are normalized to the control variant of transfection with LNPs containing DOPE (B05). Fluorescence Fluorescence LNP Cy5 mKate2 B05 1.00 0.04 1.00 0.19 B24 0.52 0.01 0.18 0.01 B25 0.42 0.03 0.12 0.02

Example 27

(103) Comparison of mRNA Transfection Using New LNPs and mRNA-LNPs Formed by Known Transfection Reagents

(104) The transfection efficiency of mRNA-LNP B05 was compared with selected known, highly efficient transfection reagents, namely D-Lin-MC3-DMA (MedChemExpress Europe) and ionisable lipidoid TT3 (Li, B.: Nano Lett. 2015, 15, 8099-8107) formulated in Example 25 as B26-B27, and also with Lipofectamine 2000 reagent (Invitrogen, used according to the standard protocol provided by the manufacturer, designated as Lip2000). Cells of the human cell line HEK293T were cultured in 96-well plates (510.sup.4 in 100 l of culture medium per well) in DMEM medium supplemented with 10% FBS at 37 C. in 5% CO.sub.2. Cells were transfected with 2 l of mRNA-LNP (with a final total concentration of all lipid components of 20 M) and subsequently incubated for 24 hours. Transfections were performed in triplicates. The fluorescence intensity of mKate2 and the percentage of cells expressing mKate2 were analysed in a BD LSR Fortessa cytometer.

(105) The new mRNA-LNPs (labelled B05) exhibited significantly higher transfection efficiency than known transfection reagents (Table 7).

(106) TABLE-US-00007 TABLE 7 Comparison of new mRNA-LNPs with mRNA-LNPs formed by commercial ionisable lipids D-Lin-MC3-DMA (B26), TT3 (B27) and Lipofectamine 2000 (Lip2000). Transfection efficiency is expressed as the percentage of cells expressing the mKate2 fluorescent protein and as the mKate2 fluorescence intensity normalized to the value from the control transfection with Lipofectamine 2000. % cells expressing Fluorescence LNP mKate2 mKate2 B05 95.20 0.84 13.29 0.02 B26 84.22 1.18 0.76 0.03 B27 90.04 2.43 1.96 0.06 Lip2000 40.70 1.13 1.00 0.03

Example 28

(107) Efficiency of mRNA Incorporation into Lipid Nanoparticles

(108) The packaging efficiency of mRNA encoding the mKate2 fluorescent protein into mRNA-LNPs B01-B27 prepared in Example 25 was determined using a Qubit 4 RNA HS Assay Kit (Life Technologies) according to the manufacturer's protocol. The efficiency of incorporation was determined by comparing the concentration of mRNA freely available in the nanoparticle solution and the concentration of mRNA released from the nanoparticles after their decomposition. mRNA-LNPs were decomposed with buffer containing Triton X-100 (10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA, 2% Triton X-100). The mRNA packaging efficiency was demonstrated, ranging from 43 to 93% (Table 8).

(109) TABLE-US-00008 TABLE 8 Efficiency of packaging mRNA encoding fluorescent protein mKate2 into mRNA-LNP, including standard deviations from triplicates. LNP mRNA packaging (%) B01 92.59 2.79 B02 82.30 2.20 B03 90.94 0.55 B04 93.18 0.77 B05 80.86 2.31 B06 83.74 0.24 B07 76.49 6.16 B08 81.22 1.33 B09 67.51 6.27 B10 78.33 0.79 B11 92.3 4.4 B12 88.3 1.0 B13 78.3 7.0 B14 60.8 2.8 B15 62.2 4.2 B16 64.3 1.4 B17 65.9 7.3 B18 65.3 3.8 B19 83.6 1.8 B20 43.4 5.8 B21 54.8 2.8 B22 66.3 9.8 B23 64.2 1.7

Example 29

(110) Cellular Toxicity of mRNA-LNP

(111) A human cell line derived from embryonic kidney cells (HEK293), the same line expressing SV40 large T antigen (HEK293T), human osteosarcoma-derived cell line (U2OS), and human hepatocyte carcinoma cell line (HepG2) were cultured in 96-well plates (510.sup.4 cells in 100 l of culture medium per well) in Dulbecco's modified medium (DMEM) or in IMDM medium (Iscove's Modified Dulbecco's medium) supplemented with 10% foetal bovine serum (FBS) at 37 C. in 5% CO.sub.2. Cells were transfected with 2 l of mRNA-LNPs generated in triplicates in Example 25 (the final total concentration of all lipid components in the well was 20 M) or 10 l of mRNA-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 analysed 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 9A,B and 10A,B.

(112) When using mRNA-LNPs with a total concentration of all lipid components of 20 M, the maximum cytotoxicity measured in the HEK293 cell line was 16%, measured for B02 and B03. No significant toxicity was demonstrated for particles B01 and B05-B23. With a 5 times higher total concentration of all lipid components of 100 M, the trend was analogous, with the highest toxicity of 38% in B03; particles B04-B08 exhibited a cytotoxicity of approximately 20%, B09 and B10 exhibited no toxicity. Particles B11-B23 also exhibited no toxicity. In the HEK293T line, the highest cytotoxicity was measured again in B02 and B03; in other particles, the toxicity was very low. Particles with total concentrations of all lipid components of 20 M exhibited almost no toxicity on the U2OS cell line, with a concentration of 100 M, the highest toxicity was 35% for B10 particles. Particles formed by lipidoids 4a, 4b, 4d and 4e were non-toxic, particles formed by lipidoids 4f, 4g, 9 and 13 were very slightly toxic (Tab. 9A, Tab. 10B).

(113) TABLE-US-00009 TABLE 9A Cytotoxicity of mRNA-LNPs expressed as cell viability (%) after the addition of 20 M transfection mixture with mRNA for individual cell line types. LNP HEK293 HEK293T U2OS Control 100.00 6.75 100.00 2.34 100.00 2.65 B01 98.15 1.00 95.54 0.29 96.51 0.47 B02 84.02 1.98 80.75 2.61 98.08 1.38 B03 84.62 0.32 75.04 2.58 90.09 1.64 B04 91.76 0.45 89.54 1.13 98.59 0.88 B05 98.98 1.73 93.75 2.51 96.45 2.97 B06 96.89 4.71 92.70 2.59 92.75 2.53 B07 96.91 5.64 93.45 0.94 92.53 2.70 B08 97.70 5.13 95.16 4.00 92.98 4.65 B09 107.46 6.15 84.34 3.78 95.89 1.87 B10 97.13 3.47 81.06 2.69 98.92 1.11

(114) TABLE-US-00010 TABLE 9B Cytotoxicity of mRNA-LNPs expressed as cell viability (%) after the addition of 20 M transfection mixture with mRNA for individual cell line types. LNP HEK293T HepG2 B11 103.8 6.8 104.2 8.4 B12 99.3 6.5 105.7 4.5 B13 101.5 5.6 104.0 7.6 B14 102.3 3.7 106.6 4.8 B15 98.9 1.4 99.7 1.9 B16 100.7 1.7 107.4 3.3 B17 100.2 4.5 108.7 4.9 B18 100.2 3.8 104.2 1.3 B19 96.6 1.6 79.6 4.2 B20 101.7 1.8 106.3 2.4 B21 105.6 1.9 106.4 1.2 B22 104.3 1.6 106.6 3.0 B23 102.9 1.1 104.7 5.0

(115) TABLE-US-00011 TABLE 10A Cytotoxicity of mRNA-LNPs expressed as cell viability (%) after the addition of 100 M transfection mixture with mRNA for individual cell line types. LNP HEK293 HEK293T U2OS Control 100.00 6.75 100.00 2.34 100.00 2.65 B01 95.98 0.88 90.01 0.24 94.63 0.58 B02 70.24 2.90 65.65 2.32 91.32 2.40 B03 62.00 2.89 57.21 2.18 75.64 4.92 B04 77.44 1.35 78.14 1.30 97.02 0.73 B05 88.00 4.82 88.92 1.39 96.51 1.87 B06 81.58 3.97 83.86 0.98 83.31 1.74 B07 87.19 1.71 91.09 1.98 85.02 1.37 B08 77.93 14.22 88.46 2.81 84.76 2.16 B09 100.44 12.84 82.46 5.08 84.88 5.59 B10 94.09 5.58 86.73 8.29 65.30 7.71

(116) TABLE-US-00012 TABLE 10B Cytotoxicity of mRNA-LNPs expressed as cell viability (%) after the addition of 100 M transfection mixture with mRNA for individual cell line types. LNP HEK293T HepG2 B11 98.2 3.3 99.7 3.8 B12 95.9 3.8 101.6 3.1 B13 90.9 4.3 96.3 3.0 B14 98.3 0.7 103.8 4.5 B15 97.5 0.6 109.9 9.6 B16 96.8 3.3 108.5 5.1 B17 96.7 2.9 105.1 6.6 B18 101.5 5.9 108.2 4.6 B19 82.2 1.8 84.4 4.2 B20 96.9 4.2 104.5 3.8 B21 96.7 1.7 102.3 6.7 B22 96.1 3.1 103.1 4.3 B23 100.1 4.4 103.6 3.9

Example 30

(117) Transfection of mRNA Using New LNPs In Vitro

(118) A human cell line derived from embryonic kidney cells (HEK293), the same line expressing SV40 large T antigen (HEK293T), liver carcinoma cells (HepG2, Huh7) and a human osteosarcoma-derived cell line (U2OS), were cultured in 96-well plates (510.sup.4 cells in 100 l culture medium per well) in Dulbecco's modified medium (DMEM) or in IMDM medium supplemented with 10% fetal bovine serum (FBS) at 37 C. in 5% CO.sub.2. Cells were transfected with 2 l of mRNA-LNP B01 to B05 prepared in Example 25 (final total concentration of all lipid components in the well was 20 M) carrying mRNA encoding the fluorescent protein mKate2 and subsequently incubated for 24 hours. 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 percentage of cells expressing the mKate2 fluorescent protein and the fluorescence intensity of the mKate2 were analyzed in a BD LSR Fortessa cytometer. For fluorescence intensity, data are normalized to the commercial transfection reagent Lipofectamine 2000.

(119) For the HEK293 line, the percentage of cells expressing the mKate2 protein was more than 2-fold higher for all the lipidoids used than for a commercially available transfection reagent. The fluorescence intensity was more than 2 higher for all lipidoids used, with the B02 particles formed by lipidoid 4d it was 3 higher than for commercially available Lipofectamine 2000. mRNA-LNPs formed with the new lipidoids transfected the HepG2 cell line in all cases at least 2.5 times better than commercial Lipofectamine 2000, the fluorescence intensity for the mRNA-LNPs B03 formed with lipidoid 4c was 4.5-fold that for Lipofectamine 2000. A similar improvement in transfection with new mRNA-LNPs compared to commercial Lipofectamine 2000 was achieved in other cell lines (Tab. 11, Tab. 12).

(120) TABLE-US-00013 TABLE 11 Transfection efficiency of new mRNA-LNPs for different cell lines expressed as a percentage of cells expressing fluorescent mKate2 protein from mRNA transfected with particles for HEK293, HepG2, HEK293T, Huh7 and U2OS lines. Statistics were evaluated by Student's unpaired t-test. The p values are always related to the control variant of Lip2000 transfection in the respective cell line; p values <0.001 are indicated with the letter a. LNP HEK293 p HepG2 p HEK293T p Huh7 p U2OS p B01 91.24 4.25 a 94.57 0.38 a 97.08 0.40 a B02 96.39 0.72 a 97.10 0.70 a 93.10 1.63 a B03 95.63 0.79 a 96.93 0.39 a 92.31 1.56 a 88.23 2.61 a 82.37 1.68 a B04 90.57 1.54 a 97.56 0.36 a 93.53 3.58 a 90.66 0.79 a B05 91.56 3.02 a 96.42 0.75 a 93.53 3.58 a 96.26 0.37 a Lip2000 42.63 17.56 a 38.10 1.54 a 46.03 1.88 a 64.67 1.40 a 53.93 1.93 a

(121) TABLE-US-00014 TABLE 12 Transfection efficiency of new mRNA-LNPs for different cell lines expressed as relative fluorescence intensity of mKate2 from mRNA transfected with particles for HEK293, HepG2, HEK293T, Huh7 and U2OS lines. For fluorescence intensity, data are normalized to the commercial transfection reagent Lipofectamine 2000. Statistics were evaluated by Student's unpaired t-test. The p values are always relative to the control variant of Lip2000 transfection in the respective cell line; values of p <0.001 are marked with the letter a, p <0.01 are marked with b. LNP HEK293 p HepG2 p HEK293T p Huh7 p U2OS p B01 2.33 0.17 a 3.05 0.22 a 2.04 0.09 a B02 2.74 0.37 a 2.87 0.17 a 3.00 0.47 a B03 2.92 0.38 a 4.54 0.22 a 3.93 0.57 a 1.19 0.08 b 2.07 0.27 a B04 3.08 0.21 a 3.93 0.24 a 3.50 0.55 a 1.26 0.08 a B05 2.61 0.11 a 3.48 0.32 a 2.87 0.31 a 1.96 0.13 a Lip2000 1.00 0.30 a 1.00 0.11 a 1.00 0.05 a 1.00 0.03 ab 1.00 0.03 a

Example 31

(122) Transfection of mRNA Using New LNPs Formed by Lipidoids Modified in Z

(123) Human cell line HepG2 was cultured in 96-well plates (510.sup.4 cells in 100 l culture medium per well) in DMEM medium supplemented with 10% FBS at 37 C. in 5% CO.sub.2. Cells were transfected with 2 l of mRNA-LNP B11-B13 prepared in Example 25 (final total concentration of all lipid components in the well was 20 M) and subsequently incubated for 24 hours. 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 percentage of cells expressing the mKate2 fluorescent protein and the fluorescence intensity of the mKate2 were analyzed in a BD LSR Fortessa cytometer. New mRNA-LNP with Z modified substituents of lipidoids were able to efficiently transfect the mRNA into HepG2 cell line (Tab. 13) in all cases better than control transfection reagent Lipofectamine 2000.

(124) TABLE-US-00015 TABLE 13 Transfection efficiency of new mRNA-LNPs expressed as a percentage of cells expressing fluorescent mKate2 protein and as relative fluorescence intensity of mKate2 from mRNA transfected with particles for HepG2 cell line. For fluorescence intensity, data are normalized to the commercial transfection reagent Lipofectamine 2000 (Lip2000). Statistics were evaluated by Student's unpaired t-test. The p values are always related to the control variant of Lip2000 transfection in the respective cell line; p values <0.001 are indicated with the letter a. % cells expressing Fluorescence LNP mKate2 p intensity p B11 97.92 0.62 a 3.81 0.51 a B12 95.92 2.55 a 1.62 0.34 a B13 95.31 0.53 a 2.23 0.26 a Lip2000 40.70 1.13 a 1.00 0.03 a

Example 32

(125) Transfection of mRNA Using New LNPs Formed by Biodegradable Lipidoids

(126) Human cell lines HEK293, HEK293T and U2OS were cultured in 96-well plates (510.sup.4 cells in 100 l culture medium per well) in DMEM or IMDM medium supplemented with 10% FBS at 37 C. in 5% CO.sub.2. Cells were transfected with 2 l of mRNA-LNP B05, B08-B10 and B14-B23 prepared in Example 25 (final total concentration of all lipid components in the well was 20 M) and subsequently incubated for 24 hours. 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 percentage of cells expressing the mKate2 fluorescent protein and the fluorescence intensity of the mKate2 were analyzed in a BD LSR Fortessa cytometer.

(127) Cell lines were transfected significantly better with the new mRNA-LNPs compared to commercial Lipofectamine 2000. Lipidoids 4e, 9, 13 and 21 had comparable effects on the percentage of cells transfected. The fluorescence intensity of the translated mRNA encoding the mKate2 protein was then always significantly increased in the HEK293 and HEK293T lines compared to commercial Lipofectamine 2000. In the U2OS line, lipidoid 13 transfected similarly to the lipidoid 4e (Tab. 14, Tab. 15).

(128) TABLE-US-00016 TABLE 14 Transfection efficiency of new mRNA-LNPs for various cell lines expressed as the percentage of cells expressing the mKate2 fluorescent protein from the mRNA transfected with the particles. HEK293, HEK293T and U2OS lines are listed. Statistics were evaluated by Student's unpaired t-test. The p values are always related to the control variant of Lip2000 transfection in the respective cell line; values of p <0.001 are marked with the letter a. LNP HEK293 p HEK293T p U2OS p B08 86.50 0.71 a 95.63 4.50 a 86.87 4.72 a B09 87.50 2.15 a 96.03 4.79 a 88.65 0.93 a B10 86.50 0.71 a 96.83 3.52 a 86.85 2.32 a B14 92.94 0.90 a B15 87.30 1.44 a B16 92.81 0.95 a B17 92.94 2.50 a B18 92.16 2.02 a B19 99.67 0.12 a B20 90.23 0.38 a B21 91.68 0.53 a B05 89.20 0.34 a 98.57 0.23 a 90.15 2.10 a Lip2000 36.73 1.86 a 59.40 2.78 a 38.87 3.18 a

(129) TABLE-US-00017 TABLE 15 Transfection efficiency of new mRNA-LNPs for various cell lines expressed as relative fluorescence intensity of mKate2 related to the Lip2000 transfection control variant. HEK293, HEK293T and U2OS lines are listed. Statistics were evaluated by Student's unpaired t-test. The p values are always related the control variant of Lip2000 transfection in the respective cell line; values of p <0.001 are marked with the letter a, p <0.01 are marked with b. LNP HEK293 p HEK293T p U2OS p B08 3.11 0.49 a 1.37 0.08 b 1.08 0.12 b B09 4.31 0.62 a 1.49 0.13 a 1.93 0.05 a B10 2.96 0.19 a 1.77 0.26 b 1.06 0.11 b B14 1.41 0.04 a B15 2.51 0.21 a B16 1.03 0.14 B17 1.74 0.29 b B18 1.94 0.08 a B19 7.47 0.90 a B05 8.54 0.44 a 2.93 0.15 a 2.14 0.10 a Lip2000 1.00 0.09 a 1.00 0.11 ab 1.00 0.15 ab

Example 33

(130) Transfection of siRNA Using New LNPs In Vitro

(131) LNPs containing small interfering RNA (siRNA, catalog number AM4626, Ambion) causing degradation of mRNA encoding green fluorescent protein (GFP) were prepared as follows: 300 l of A28 transfection reagent solution prepared in Example 24 was mixed with a solution of 1.20 nmol siRNA in 300 l 10 mM citrate buffer (pH 3.0) using a microfluidic device analogously to Example 25 The resulting siRNA-LNPs were immediately diluted in 600 l PBS; the corresponding nanoparticles labeled B28 were thus formed from transfection reagent A28. LNPs carrying control (scrambled; 4390843, Ambion) siRNAs that do not target any endogenously cell-transcribed mRNA (B29) were prepared analogously. Lipofectamine RNAiMax (Invitrogen) was used as a control transfection reagent specifically for siRNA transfection according to the manufacturer's standard protocol. A human cell line U2OS stably expressing the green fluorescent protein (GFP) was used for siRNA-LNP knockdown. Cells were cultured in 96-well plates (510.sup.4 cells in 100 l culture medium per well) in DMEM medium supplemented with 10% FBS at 37 C. in 5% CO.sub.2. Cells were transfected with 2 l of siRNA-LNP, the final total concentration of all lipid components in the well was 20 M; final siRNA concentration was 16 nM) and subsequently incubated for 24 hours. Transfections were performed in three biological replicates. The percentage of cells expressing the green fluorescent protein GFP and the fluorescence intensity of the GFP were analyzed in a BD LSR Fortessa cytometer.

(132) With the new siRNA-LNPs (B28), a decrease in GFP expression was observed down to 1% of GFP-expressing cells; with commercial RNAiMax, GFP expression was still detected in 13.6% of cells. The fluorescence intensity of GFP when using the new siRNA-LNPs (B28) decreased by 1.25-fold compared to the commercial RNAiMax reagent (Tab. 16).

(133) TABLE-US-00018 TABLE 16 Reduction in GFP-encoding mRNA in the U2OS cell line by siRNA- LNP (B28) expressed as a percentage of GFP-expressing cells and GFP fluorescence intensity compared to the commercial transfection reagent RNAiMAx and siRNA-LNP carrying control siRNA (B29). Statistics were evaluated by Student's unpaired t-test. The p values are related to the B28 transfection mixture tested; values of p <0.001 are marked with the letter a, p <0.01 are marked with b. % cells expressing GFP fluorescence LNP GFP p intensity p Control 82.30 1.60 0.86 0.02 B28 1.00 0.30 a 0.26 0.03 ab B29 83.60 0.90 a 0.92 0.06 a RNAiMax 13.60 1.20 a 0.32 0.03 b

(134) siRNA-LNPs containing small interfering RNA (siRNA, catalog number 4392420, Ambion) causing the degradation of mRNA encoding tyrosyl-DNA phosphodiesterase 2 (TDP2) were prepared in the same manner as B28 particles in this example. The corresponding nanoparticles designated B30 were thus formed from the transfection reagent A28. LNPs carrying control (scrambled; 4390843, Ambion) siRNAs that do not target any endogenously cell-transcribed mRNA (B29) were also used as a control. Lipofectamine RNAiMax (Invitrogen) was used as a control transfection reagent specifically for siRNA transfection. The human cell line HEK293 and two lines derived from human multiple myeloma, which are very difficult to transfect with available transfection reagents, were used for siRNA-LNP knockdown (Brito J. L. R., Brown N., Morgan G. J. (2010) Transfection of siRNAs in Multiple Myeloma Cell Lines. In: Min W P., Ichim T. (eds) RNA Interference. Methods in Molecular Biology (Methods and Protocols), vol 623. Humana Press). Cells were cultured in 96-well plates (510.sup.4 cells in 100 l culture medium per well) in DMEM medium supplemented with 10% FBS at 37 C. in 5% CO.sub.2. Cells were transfected with 2 l siRNA-LNP (with a final total concentration of all lipid components of 20 M and a final concentration of siRNA of 16 nM) and subsequently incubated for 24 hours. Transfections were performed in three biological replicates. RNA was isolated with an RNAeasy Plus Micro Kit (Qiagen). The cDNA was prepared with TATAA GrandScript cDNA Supermix (TATAAbiocenter) according to the manufacturer's recommendations. Quantitative RT-PCR was performed in 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)).

(135) In all these cases, the new siRNA-LNPs significantly reduced the level of TDP2 mRNA in cells compared to the commercial transfection reagent RNAiMax. In the HEK293 cell line it was 2.86-fold, in the OPM-2 myeloma line 4.3-fold, and in the RPMI8226 myeloma line 6.7-fold compared to a commercial reagent for siRNA transfection (Tab. 17).

(136) TABLE-US-00019 TABLE 17 Reduction of endogenously expressed TDP2 mRNA levels in cells by new siRNA-LNPs (B30) compared to the commercial transfection reagent RNAiMax and compared to control siRNA-LNPs (B29) in the HEK293, OPM-2 and RPMI8226 cell lines. Statistics were evaluated by Student's unpaired t-test. The p values are related to the B30 transfection mixture tested; p values <0.001 are indicated with the letter a. LNP HEK293 p OPM-2 p RPMI8226 p Control 1.00 0.10 1.00 0.01 1.00 0.03 B30 0.03 0.01 a 0.14 0.02 a 0.13 0.00 a B29 0.99 0.17 a 1.09 0.01 a 0.78 0.01 a RNAiMax 0.09 0.01 a 0.58 0.02 a 0.87 0.11 a

Example 34

(137) Transfection of Plasmid DNA Using New LNPs In Vitro

(138) LNPs containing 4706 bp plasmid DNA (Evrogen, Cat. No. FP181) encoding the mKate2 fluorescent protein were prepared as follows: 300 l of the A28 transfection reagent solution prepared in Example 24 was mixed with a solution of 120 g of plasmid DNA in 300 l of 10 mM citrate buffer (pH 3.0) using a microfluidic device analogously to Example 25. The resulting DNA-LNPs were immediately diluted in 600 l PBS; the corresponding nanoparticles designated B31 were thus formed from transfection reagent A28. Transfections were performed on the human cell line HEK293T. Cells were cultured in 96-well plates (510.sup.4 cells in 100 l culture medium per well) in DMEM medium supplemented with 10% FBS at 37 C. in 5% CO.sub.2. Cells were transfected with 2 l of DNA-LNP (with a final total concentration of all lipid components of 20 M) and subsequently incubated for 24 hours. Lipofectamine 2000 (Lip2000, Invitrogen) was used as a control transfection reagent. Transfections were performed in three biological replicates, with each biological replicate having three technical replicates. The percentage of cells expressing the mKate2 fluorescent protein and the fluorescence intensity of the mKate2 were analyzed in a BD LSR Fortessa cytometer.

(139) The percentage of cells expressing the fluorescent protein mKate2 was 3.8-fold higher with the new DNA-LNPs compared to the commercial transfection reagent. The fluorescence intensity was 2.9 times higher than with the commercial reagent (Tab. 18).

(140) TABLE-US-00020 TABLE 18 Transfection efficiency of new DNA-LNPs (B31) compared to commercial Lipofectamine 2000 (Lip2000) in the HEK293T cell line expressed as % of mKate2-expressing cells and as mKate2 fluorescence intensity. For fluorescence intensity, data are normalized to the commercial transfection reagent Lipofectamine 2000. Statistics were evaluated by Student's unpaired t-test. Values of p <0.001 are indicated with the letter a. % cells expressing mKate2 fluorescence LNP mKate2 p intensity p B31 91.16 0.98 a 2.93 0.36 a Lip2000 24.30 1.00 a 1.00 0.07 a

Example 35

(141) Transfection of mRNA with New LNPs into Human Primary Hepatocytes

(142) The gene encoding the NanoLuc bioluminescent protein was amplified from plasmid pET51b(+)_S-Luc_CLIP (Addgene, Cat. No. 113923; obtained as a gift from Kai Johnsson) using the primers: (5-TAATACGACTCACTATAGGG-3 (SEQ ID NO. 7); 5-GCTAGTTATTGCTCAGCGG-3 (SEQ ID NO. 8)). Messenger RNA (mRNA) was prepared in vitro analogously to Example 25 and packaged into LNP as follows: A 300 l sample of transfection reagent A28, 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 25. The resulting mRNA-LNPs were immediately diluted in 600 l PBS; the corresponding nanoparticles labeled B32 were thus formed from transfection reagent A28. Primary hepatocytes were isolated from a human donor according to a published protocol (LeCluyse, E. L.: Methods in Molecular Biology 2005; 290, 207-230). Cells were transfected with 2 l of mRNA-LNP (with a final total concentration of all lipid components of 20 M) and incubated for 24 hours. Lipofectamine 2000 transfection reagent was used as a control. Substrate in the Nano-Glo Luciferase Assay System kit (Promega) was then added to the cells and the luminescence intensity was analyzed in a Microplate Reader: Infinite M1000 PRO (Tecan).

(143) The new mRNA-LNPs efficiently transfected human primary hepatocytes, with a significant 2.0-fold increase in luminescence compared to the non-transfected control (Tab. 19).

(144) TABLE-US-00021 TABLE 19 Transfection efficiency of new mRNA-LNPs (B32) in the primary line of human hepatocytes compared to the commercial transfection reagent Lipofectamine 2000 expressed as bioluminescence intensity normalized to the value from control transfection. Statistics were evaluated by Student's unpaired t-test. Values of p <0.05 are indicated with the letter c). LNP bioluminescence intensity p B32 1.97 0.60 c Lip2000 1.00 0.12 c

Example 36

(145) Transfection of Cyclic Dinucleotides with New LNPs In Vitro

(146) The cyclic dinucleotide (2,3-cGAMP) (Sigma, cat. No. SML1229-0.5UMO) was packaged in LNP as follows: 300 l samples of A01-A06 transfection reagents, and 120 nmol of cGAMP in 300 l of 10 mM citrate buffer (pH 3.0) were added to the LNP using a microfluidic device analogous to Example 25. The resulting cGAMP-LNPs were immediately diluted in 600 l PBS; samples of nanoparticles designated B33-B38 were thus formed from the corresponding transfection reagents A01-A06. A reporter assay showing the degree of induction of the interferon response by the cyclic dinucleotide cGAMP depending on the STING pathway was used to analyse the efficiency of cGAMP-LNP particle transfection. For this purpose, a cell reporter line HEK293T expressing the common type of STING protein and a luciferase reporter gene under the IRF3 interferon-stimulated (ISRE) promoter were used according to Novotn et al. (Novotn, B.: J. Med. Chem. 2019, 62 (23), 10676-10690). Cells were transferred to poly-D-lysine (Sigma-Aldrich)-coated 96-well plates (Greiner Bio-One) at a density of 2.510.sup.4 in DMEM medium containing glucose (containing L-glutamine; Biowest) supplemented with 10% FBS (Capricorn Scientific) and 1% penicillin-streptomycin (Biowest). After incubation at 37 C. in 5% CO.sub.2 overnight, serially diluted compounds were added to the cells for 7 hours. In parallel, HEK293T cells were incubated with the test compounds alone for 30 minutes, washed twice with fresh medium, and then cultured for an additional 6.5 hours. Finally, 50 l of cell culture medium was mixed with 30 l of Bright-Glo Luciferase Assay System reagent (Promega) in white 96-well plates and luminescence was read in a Spark spectrophotometer (TECAN, Grodig). Values of 50% effective concentration (EC.sub.50) were calculated using GraphPad Prism (La Jolla) as described in Novotn et al. (Novotn, B.: J. Med. Chem. 2019, 62 (23), 10676-10690) (Tab. 20).

(147) The cyclic dinucleotide 2,3cGAMP exhibited EC.sub.50 values of STING activation30 M. All new cGAMP-LNPs formed by ionizable lipidoids 4a-4f efficiently transfected HEK293T cells and activated STING in the nanomolar region, increasing transfection efficiency approximately 2000-15000. The most effective was lipidoid 4d, exhibiting an EC.sub.50 of 2.000.36 nM.

(148) TABLE-US-00022 TABLE 20 EC.sub.50 values of STING activation for 2,3-cGAMP transfected with new cGAMP-LNPs (B33-B38) formed by ionizable lipidoids 4a-4f. LNP EC.sub.50 STING activation (nM) B33 9.00 1.75 B34 12.00 1.80 B35 10.00 6.42 B36 2.00 0.36 B37 4.00 1.12 B38 14.00 2.61

Example 37

(149) Toxicity of New mRNA-LNPs In Vivo

(150) Messenger RNA (mRNA) encoding NanoLuc protein was prepared and packaged into particles analogously to Example 35. The resulting mRNA-LNPs (B32) were administered intraperitoneally to three C57Bl/6 mice (BIOCEV, Vestec) at a concentration of 0.5 mg mRNA/kg, wherein three control C57Bl/6 mice were administered PBS intraperitoneally. Another three C57Bl/6 mice were administered mRNA-LNP at a 5-fold higher concentration (2.5 mg mRNA/kg) in the same manner. Similarly, identical doses of mRNA-LNPs were again administered to 3 C57Bl/6 mice (0.5 mg mRNA/kg) and 3 C57Bl/6 mice (2.5 mg mRNA/kg) intravenously, and the animals were anesthetized prior to the administration (2.5 mg/mouse ketaminum, 0.4 mg/mouse xylazinum, Bioveta). Mice were then observed for 48 hours and none showed any signs of toxicity or phenotypic changes compared to control animals.

Example 38

(151) Biodistribution of New mRNA-LNPs In Vivo

(152) The gene encoding the CRE recombinase protein was amplified from the plasmid pCAG-Cre-IRES2-GFP (Addgene, catalog number 26646, donated by Anjen Chenn) using the primers: (5-TAATACGACTCACTATAGAATTTACT-3 (SEQ ID NO. 9); 5-CTAATCGCCATCTTCCAGCA-3 (SEQ ID NO. 10)). Messenger RNA (mRNA) was prepared in vitro analogously to Example 25 and packaged into LNP as follows: A 300 l sample of transfection reagent A28, 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 25. The resulting mRNA-LNPs were immediately diluted in 600 l PBS; the corresponding nanoparticles labeled B39 were thus formed from transfection reagent A28. mRNA-LNPs (B39) were administered intravenously at a concentration of 0.5 mg mRNA/kg to 2.5 mg mRNA/kg in each case to 3 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 the cells to which mRNA-LNPs carrying Cre recombinase mRNA were successfully delivered, chromosomal recombination and subsequent excision of the membrane red protein gene (so-called red tomato) and turning on the transcription of membrane green protein (GFP) gene occurred. Mice, including non-particulate control mice, were sacrificed 3 days after particulate application, and all organs were subjected to histological analysis according to a standardized protocol. Histological images show a complete distribution of particles into the liver, which led to a 30-50% conversion of cells expressing the red membrane protein to cells expressing the green membrane protein 3 days after application (FIG. 7). In FIG. 7, histological images of liver show the conversion of cells expressing the red membrane protein to cells expressing the green membrane protein 3 days after application of the mRNA-LNPs [2.5 mg/kg of mRNA] encoding Cre recombinase (labeled as 3 dpi mRNA-LNP). PBS control-injection of PBS buffer only.

Example 39

(153) Stability of New mRNA-LNPs and siRNA-LNPs at 4 C.

(154) Human cell line HEK293T was cultured in 96-well plates (510.sup.4 cells in 100 l culture medium per well) in DMEM medium supplemented with 10% FBS at 37 C. in 5% CO.sub.2. Cells were transfected with 2 l of mRNA-LNP B05, prepared in Example 25 (final total concentration of all lipid components in the well was 20 M) and subsequently incubated for 24 hours. The B05 mRNA-LNPs were stored at 4 C. and transfections were analogously repeated one, three and five weeks after assembly. Transfections were performed in triplicates. The fluorescence intensity of the mKate2 was analyzed in a BD LSR Fortessa cytometer.

(155) Transfection efficiency of the new mRNA-LNPs (labelled B05) remained unchanged for at least three weeks and decreased to 81% compared to Day 1, while stored at 4 C. (Tab. 21).

(156) TABLE-US-00023 TABLE 21 Transfection efficiency of B05 mRNA-LNPs stored at 4 C. for HEK293 cells expressed as relative fluorescence intensity of mKate2. Data normalized to fluorescence intensity measured at Day 1 after assembly. Five weeks later, the B05 mRNA-LNPs still kept 81% of transfection efficiency while stored at 4 C. Time Fluorescence of mKate2 1 day 1.00 0.17 1 week 1.14 0.26 3 weeks 1.04 0.16 5 weeks 0.81 0.11

(157) siRNA-LNPs (labelled B30 and B40) containing small interfering RNA (siRNA, catalog number 4392420, Ambion) causing the degradation of mRNA encoding tyrosyl-DNA phosphodiesterase 2 (TDP2) were prepared in the same manner as described in Example 33 and were stored at 4 C. siRNA-LNPs B40 were formed from transfection reagent A29 prepared in Example 24. HEK293T cells were cultured in 96-well plates (510.sup.4 cells in 100 l culture medium per well) in DMEM medium supplemented with 10% FBS at 37 C. in 5% CO.sub.2. Cells were transfected with 2 l siRNA-LNPs (with a final total concentration of all lipid components of 20 M and a final concentration of siRNA of 16 nM) and subsequently incubated for 24 hours. Transfections were performed in triplicates. RNA isolation, cDNA preparation and qRT-PCR were performed as described in Example 33. The whole procedure was repeated one, two and three months after the LNP assembly.

(158) The transfection efficacy of the new siRNA-LNPs (labelled B30 and B40) expressed as their ability to reduce the level of TDP2 mRNA in cells remained practically unchange while stored at 4 C. for three months (Tab. 22).

(159) TABLE-US-00024 TABLE 22 Relative level of TDP2 mRNA in HEK293T cells after transfection of siRNA-LNPs stored at 4 C. at respective time points. Time B30 B40 1 day 0.0380 0.0107 0.0336 0.0079 1 month 0.0700 0.0036 0.0817 0.0100 2 months 0.0423 0.0026 0.0492 0.0037 3 months 0.0387 0.0067 0.0646 0.0164

Example 40

(160) Transfection of siRNA by Preformed Empty LNPs

(161) Transfection reagents A28 and A29 from Example 24 were used to assemble empty nanoparticles B41 and B42 in citrate buffer excluding nucleic acid analogously to Example 25, with no post-dilution. One l of LNPs was mixed with 1 pmol of siRNA targeting TDP2 10 minutes prior to transfection, analogously as recommended for Lipofectamine RNAiMax, and incubated at room temperature. HEK293T and HepG2 cells (510.sup.4 cells in 100 l culture medium per well) were transfected with the mixture of preassembled LNPs and siRNA and incubated for 24 hours. Transfections were performed in triplicates. Lipofectamine RNAiMax was used as a control transfection reagent. Preassembled LNPs subsequently mixed with 1 pmol of siRNA were able to knock down TDP2 mRNA expression by 90-97%, both significantly better then commercial transfection reagent Lipofectamine RNAiMax (Tab. 23).

(162) TABLE-US-00025 TABLE 23 Reduction of TDP2 mRNA levels in cells by preassembled empty LNPs mixed with siRNA targeting TDP2 compared to commercial transfection reagent RNAiMax. Statistics were determined by Student's unpaired t-test. P values are related to control transfection with Lipofectamine RNAiMax, values p <0.001 are indicated by letter a, values p <0.01 are indicated by letter b. LNP HEK293T p HepG2 p Control 1.00 0.25 1.00 0.08 B41 0.03 0.01 a 0.10 0.03 b B42 0.07 0.01 b 0.08 0.01 a RNAiMax 0.12 0.03 ab 0.18 0.01 ab

Example 41

(163) Transfection of Peripheral Blood Mononuclear Cells (PBMC) by siRNA-LNPs and Evaluation of Cytokine Response

(164) Transfection reagent A28 from Example 24 was used to form siRNA-LNP designated B43 containing siRNA causing the degradation of mRNA encoding Poly(U)-binding-splicing factor (PUF60). The siRNA-LNPs were prepared analogously as in Example 33. Lipofectanime RNAiMax was used as a control transfection reagent. Peripheral blood mononuclear cells (PBMC) from 3 anonymous blood donors (with the agreement number 13/06/2012 of ethical committee of the Institute of Hematology and Blood Transfusion, Prague, Czech Republic) were isolated by Ficoll (Ficoll Paque Plus, 17-1440-02, GE Healthcare) gradient and cultured in RPMI medium supplemented with 10% of FBS and 50 U/ml of penicillin/streptomycin. Cells were seeded at 10.sup.5 cells/100 ul (200 ul in total) in 96-well plates and incubated at 37 C. The ON-target plus SMARTpool siRNA targeting PUF60 were purchased from Dharmacon (catalogue number L-012505-01-0005; llkirch, France) and resuspended in water at 20 pmol/ul. The final concentrations of 1 pmol or 10 pmol were used. Each condition was performed in triplicate. 24 hours after transfection the cells were harvested including the supernatant. Total RNA was extracted using Nucleospin RNA extraction kit (Macherey Nagel) following manufacture's instruction. Reverse transcription was performed with 500 ng of RNA. Quantitative RT-PCR was performed in a LightCycler 480 (Roche Life Science) in duplicate using the SYBERGREEN mix. The primers for amplifying PUF60 were as follows: 5-CCTTCAACCGCATCTACGTG-3 (SEQ ID NO. 7) and 5-CTGGGCCTTCTCGTACTCAA-3 (SEQ ID NO. 8). RPLPO was used to normalize the data (Primers: 5-CACCATTGAAATCCTGAGTGATG-3 (SEQ NO. 9) and 5-TGACCAGCCCAAAGGAGAAG-3 (SEQ NO. 10)). The quantities of total IFN- and IFN- produced by PBMC after transfections were measured in cell-free supernatants using human ELISA kits (Human IFN- ELISABASIC kit (HRP), 3425-1H-20, Mabtech and Human IFN-1 ELISABASIC kit, 3570-1H-20, Mabtech). 24 hours treatment of PBMC with 1 M CpG ODN 2216 Class A (Invivogen) was used as a positive control.

(165) In all cases, the siRNA-LNPs reduced the level of PUF60 mRNA in PBMC cells (Tab. 24) without upregulating the cytokine response as opposed to RNAiMax (Tab. 25 and Tab. 26).

(166) TABLE-US-00026 TABLE 24 Reduction of endogenously expressed PUF60 mRNA levels in PBMC by new siRNA-LNPs (B43) compared to the transfection reagent RNAiMax and compared to non-treated cells. LNP Donor 1 Donor 2 Donor 3 Non treated 1.00 0.23 1.00 0.34 1.00 0.18 B43 [1 pmol of siRNA] 0.55 0.04 0.12 0.20 0.65 0.32 B43 [10 pmol of siRNA] 0.40 0.14 0.50 0.02 0.07 0.03 RNAiMax [1 pmol of siRNA] 0.51 0.08 1.15 0.01 0.62 0.40 RNAiMax [10 pmol of 0.21 0.20 0.29 0.30 0.22 0.10 siRNA]

(167) TABLE-US-00027 TABLE 25 IFN- response of PBMC after treatment of siRNA-LNPs compared to RNAiMax. LNP Donor 1 Donor 2 Donor 3 Non treated 2.38 2.06 0.44 0.56 0.00 0.00 Positive control 533 13 661 58 2265 29 B43 [1 pmol of siRNA] 0.00 0.00 1.52 1.54 0.00 0.00 B43 [10 pmol of siRNA] 0.00 0.00 0.00 0.00 16.8 20.1 RNAiMax [1 pmol of siRNA] 0.00 0.00 6.05 8.58 0.65 1.12 RNAiMax [10 pmol of 433 251 1814 243 1951 31 siRNA]

(168) TABLE-US-00028 TABLE 26 IFN- response of PBMC after treatment of siRNA-LNPs compared to RNAiMax. LNP Donor 1 Donor 2 Donor 3 Non treated 2.49 2.34 2.99 2.75 37.9 7.4 Positive control 713 67 1765 180 1000 52 B43 [1 pmol of siRNA] 0.00 0.00 0.00 0.00 20.4 2.7 B43 [10 pmol od siRNA] 11.7 20.2 0.00 0.00 40.7 4.2 RNAiMax [1 pmol of siRNA] 0.00 0.00 0.00 0.00 44.5 13.2 RNAiMax [10 pmol of 28.7 49.8 100 59 706 188 siRNA]

Example 42

(169) Effectivity of siRNA-LNPs In Vivo

(170) Transfection reagent A29 from Example 24 was used to form siRNA-LNPs designated as B44 with siRNA targeting mouse apolipoprotein B (ApoB) gene, a hepatocyte-expressed gene involved in cholesterol transport (ApoB) (catalogue number 238055 Apob mouse siPOOL-40 kit, siTOOLs Biotech GmbH) and alternatively siRNA-LNPs (B45) with control non-targeted siRNA-LNPs (enclosed in 238055 Apob mouse siPOOL-40 kit, siTOOLs Biotech GmbH), assembled as described in Example 33. The siRNA-LNPs were dialyzed to PBS. The 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 5 C57Bl/6 mice (BIOCEV, Czech Center of Phenogenomics, Vestec) at a concentration of 32 g of siRNA and 16 g of siRNA, respectively, wherein the control 5 mice were administered with 32 g of non-targeting siRNA-LNPs and another 5 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.

(171) 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. 27).

(172) TABLE-US-00029 TABLE 27 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 B45 LNP with control non-targeted siRNA; values of p <0.001 are marked with the letter a, p <0.01 are marked with b. LNP Total cholesterol p1 p2 Triglycerides p1 p2 LDL-C p1 p2 Control 1.99 0.34 a 0.46 0.12 ab 0.39 0.04 a B44 [16 g] 0.43 0.18 a a 0.18 0.03 a a 0.15 0.05 a a B44 [32 g] 0.35 0.13 a a 0.19 0.03 b a 0.13 0.03 a a B45 [32 g] 2.58 0.61 a 0.53 0.04 a 0.84 0.12 a

Example 43

(173) Effectivity of Nucleoside-Modified mRNA-LNPs In Vivo

(174) The gene encoding the CRE recombinase protein was amplified from the plasmid pCAG-Cre-IRES2-GFP (Addgene, catalog number 26646, donated by Anjen Chenn) using the primers: (5-TAATACGACTCACTATAGAATTTACT-3 (SEQ ID NO. 9); 5-CTAATCGCCATCTTCCAGCA-3 (SEQ ID NO. 10)). Messenger RNA (mRNA) was prepared in vitro analogously to Example 25, with the exception that CTP was 100% exchanged for 5-Methyl-CTP (NU-1138L, Biogen Praha s.r.o.) and UTP was 100% exchanged for N1-Methylpseudo-UTP (NU-890L, Biogen Praha s.r.o.), and packaged into LNP as follows: A 300 l sample of transfection reagent A28, 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 25. The resulting mRNA-LNPs were immediately diluted in 600 l PBS; the corresponding nanoparticles labeled B46 were thus formed from transfection reagent A28. mRNA-LNPs (B46) were administered intravenously at a concentration of 0.5 mg mRNA/kg to 2.5 mg mRNA/kg in each case to 3 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 the cells to which mRNA-LNPs carrying Cre recombinase mRNA were successfully delivered, chromosomal recombination and subsequent excision of the membrane red protein gene (so-called red tomato) and turning on the transcription of membrane green protein (GFP) gene occurred. Mice, including non-particulate control mice, were sacrificed 3 days after particulate application, and all organs were subjected to histological analysis according to a standardized protocol. Histological images show a complete distribution of particles into the liver, which led to a 35-75% conversion of cells expressing the red membrane protein to cells expressing the green membrane protein 3 days after application.