Cyclen-Based Cationic Lipids for Gene Delivery
0.68 (s, 3H, cholesterol-H), 0.85–2.02 (br, 43H, cholesterol-H and
N(CH2CH3)2), 2.32 (d, 2H, cholesterol-H), 2.66 (m, J = 21.2 Hz, 4H,
N(CH2CH3)2), 3.30 (s, 2H, NCH2CO), 4.67 (m, 1H, cholesterol-H), 5.35
(s, 1H, C=CH-). 13C NMR (CDCl3, 100 MHz): 21.02, 22.56, 22.82,
23.83, 24.28, 27.82, 28.01, 28.23, 31.84, 31.89, 35.79, 36.17, 36.58,
36.97, 38.15, 39.51, 39.72, 42.30, 47.73, 50.01, 54.42, 56.12, 56.67,
74.02, 76.81, 77.02, 77.24, 122.70, 139.57, 170.92. ESI MS:
500.45(M+H+).
49.84, 55.25, 55.75, 56.36, 58.16, 62.03, 66.80, 77.31, 79.54, 80.73,
109.26, 123.24, 132.57, 138.74, 164.31. ESI MS: 1103.63 (M++H).
General process for the preparation of L1 and
L2
Precursor (5a and 5b) (1.2 mmol) was suspended in anhydrous di-
chloromethane (15 mL), then to the resulting solution was added
dropwise a solution of trifluoroacetic acid (5 mL) in anhydrous di-
chloromethane (5 mL) under ice-cold water and N2 atmosphere. And
then, the obtained mixture was stirred at room temperature for 6 h.
The solvent and excess trifluoroacetic acid were removed under
reduced pressure to obtain yellow residue. Then, under stirring,
anhydrous ethyl ether was added dropwise into the residue to give
the final lipids as a white solid in 98–100% yield.
Compound 3b: IR (per cm): 3423, 2941, 2896, 2848, 1724, 1454,
1
1377, 1271, 1244, 1141, 1097, 1052, 1010, 982, 959, 899, 867. H
NMR (CDCl3, 400 MHz): d 0.79 (t, J = 6 Hz, 6H, diosgenin-H), 0.96–
2.02 (br, 32H, diosgenin-H and N(CH2CH3)2), 2.33 (d, 2H, diosgenin-
H), 2.65 (q, J = 14.4 Hz, 4H, N(CH2CH3)2), 3.30 (s, 2H, NCH2COO),
3.37 (t, 1H, diosgenin-H), 3.40 (t, 1H, diosgenin-H), 4.40 (m, 1H,
diosgenin-H), 4.75 (m, 3H, diosgenin-H), 5.38 (s, 1H, C=CH-). 13C
NMR (CDCl3, 100 MHz): 16.27, 17.13, 19.33, 20.79, 27.78, 28.79,
30.28, 31.38, 31.82, 32.02, 36.71, 36.92, 38.11, 39.70, 40.23, 41.59,
47.72, 49.90, 54.37, 56.40, 62.06, 66.81, 73.90, 80.78, 109.24,
122.40, 139.59, 170.89. ESI MS: 528.69 (M+H+).
Cationic lipid L1: IR (per cm): 3411, 2947, 2867, 1740, 1687, 1464,
1
1201, 1174, 1129, 827, 799, 719. H NMR (DMSO-d6, 400 MHz): d
0.66 (s, 3H, cholesterol-H), 0.84–1.99 (br, 47H, cholesterol-H,
N(CH2CH3)2), 2.36 (d, 2H, cholesterol-H), 2.75 (s, 4H, cyclen-H), 2.85
(s, 4H, cyclen-H), 3.06 (s, 4H, cyclen-H), 3.14 (m, 4H, cyclen-H), 3.47
(s, 4H, N(CH2CH3)2), 4.24 (s, 2H, benzene-CH2-cyclen), 4.65 (s, 1H,
cholesterol-H), 4.70 (s, 2H, benzene-CH2-N(CH2CH3)2), 5.40 (s, 1H,
C=CH-), 5.76 (s, 2H, (CH3CH2)2N-CH2-COO), 7.49 (m, 4H, benzene-H).
13C NMR (DMSO-d6, 100 MHz): 22.85, 23.12, 23.66, 24.31, 27.48,
27.86, 28.25, 31.14, 31.81, 35.66, 36.12, 36.53, 36.75, 37.66, 45.11,
47.78, 49.84, 54.94, 55.38, 55.81, 56.04, 56.48, 56.57, 62.04, 76.23,
123.17, 126.81, 131.06, 133.17, 138.62, 139.38, 164.89. HR MS
General process for the preparation of 5
A solution of diethyl amine derivatives (3a and 3b) (6 mmol) and 1-
bromomethyl4-(4, 7, 10-tris-(tertbutyloxycarbonyl)-1, 4, 7, 10-tetraaza-
cyclododecane-1-yl-methyl)benzene (4) (7.9 g, 12 mmol) in anhydrous
acetonitrile (30 mL) was refluxed at 90 ꢀC over a period of 60–96 h,
after which TLC indicated the disappearance of the starting material
diethyl amine derivatives. Then, reaction mixture was cooled and the
acetonitril was rotary-evaporated to furnish a yellow residue, and it
was purified by chromatography over silica (dichloromethane ⁄ metha-
nol, 16:1) to obtain white foamy solid in 45–60% yield.
+
(C49H84N5O2 ): Calcd. 774.6625; Found. 774.6564.
Cationic lipid L2: IR (per cm): 3432, 2949, 1743, 1683, 1457, 1203,
1177, 1137, 1055, 1008, 984, 899, 799, 720. 1H NMR (CDCl3,
400 MHz): d 0.79 (s, 6H, diosgenin-H), 0.97–2.00 (br, 38H, diosge-
nin-H and NCH2CH3), 2.37 (s, 2H, diosgenin-H), 2.87–3.18 (br, 16H,
cyclen-H), 3.35–3.40 (t, J = 21.6, 1H, diosgenin-H), 3.46–3.49 (t,
J = 14.0, 1H, diosgenin-H), 3.57 (s, 4H, N(CH2CH3)2), 4.03 (s, 2H,
benzene-CH2-Cyclen), 4.40 (m, 1H, diosgenin-H), 4.75 (s, 3H, ben-
zene-CH2-N(CH2CH3)2 and C=CH-), 5.42 (s, 2H, NCH2COO), 7.44–7.46
(m, 4H, benzene-H). 13C NMR (DMSO-d6, 100 MHz): 30.26, 31.13,
31.35, 31.89, 31.96, 36.68, 36.73, 37.65, 39.54, 39.68, 39.82, 39.96,
40.10, 40.24, 40.38, 41.55, 42.47, 42.59, 45.03, 47.73, 49.79, 54.94,
55.66, 55.79, 56.15, 62.07, 62.24, 66.37, 76.21, 79.68, 80.63,
108.88, 116.23, 118.20, 122.99, 126.82, 131.08, 133.16, 138.56,
Compound 5a: IR (per cm): 3426, 2941, 1743, 1691, 1462, 1414,
1365, 1249, 1218, 1172, 1029, 979, 861, 773. 1H NMR (CDCl3,
400 MHz): d 0.68 (s, 3H, cholesterol-H), 0.86–2.05 (br, 72H, choles-
terol-H, N(CH2CH3)2 and Boc-H), 2.01–2.65 (br, 6H, cholesterol-H and
cyclen-H), 3.32–3.69 (br, 12H, cyclen-H), 3.80–3.86 (br, 4H,
N(CH2CH3)2), 4.44 (s, 2H, Ar-CH2-cyclen), 4.72 (m, 1H, cholesterol-H),
5.04 (s, 2H, Ar-CH2-N(CH2CH3)2), 5.31 (s, 2H, Et2NCH2COO), 7.33–
7.36 (m, 2H, benzene-H), 7.50–7.52 (m, 2H, benzene-H). 13C NMR
(CDCl3, 100 MHz): 24.21, 27.48, 27.92, 27.99, 28.13, 28.43, 28.62,
31.19, 31.77, 31.84, 35.70, 36.13, 36.51, 36.77, 37.69, 39.45, 39.66,
42.27, 47.72, 49.96, 54.24, 55.34, 55.81, 56.12, 56.63, 63.06, 76.82,
79.48, 123.49, 125.68, 125.74, 127.79, 130.98, 132.59, 133.91,
138.69, 155.36, 155.85, 164.26. ESI MS: 1074.43 (M+).
+
139.42, 158.80, 159.02, 164.88. HR MS (C49H80N5O5 ): Calcd.
802.6210; Found. 802.6207.
Compound 5b: IR (per cm): 3416, 2951, 3951, 1742, 1691, 1560,
1414, 1367, 1248, 1218, 1172, 1051, 982, 898, 864, 774. H NMR
Preparation of cationic liposome
1
Individual cationic lipid (0.005 mmol) or its mixture with DOPE in
the desired mole ratio was dissolved in anhydrous chloroform
(5 mL) in autoclaved glass vials. Thin films were made by slowly
rotary-evaporating the solvent at room temperature. Last traces
of organic solvent were removed by keeping these films under
vacuum above 8 h. The dried films and freshly autoclaved Tris
buffer (10 mM, PH 7.4) were preheated to 70 ꢀC, then the Tris
buffer (5 mL) was added to the films such that the final concen-
tration of the cationic lipid was 1 mM. The mixtures were vor-
texed vigorously until the films were completely resuspended.
(CDCl3, 400 MHz): d 0.79 (s, 6H, diosgenin-H), 0.97–2.05 (br, 60H,
diosgenin-H, Boc-H and NCH2CH3), 2.38 (s, 2H, diosgenin-H), 2.65
(br, 4H, cyclen-H), 3.32–3.57 (br, 14H, cyclen-H and diosgenin-H),
3.70–3.85 (br, 6H, N(CH2CH3)2 and benzene-CH2-cyclen), 4.41–4.42
(br, 3H, diosgenin-H and NCH2COO), 4.75 (m, 1H, diosgenin-H), 5.03
(m, 2H, benzene-CH2-N(CH2CH3)), 5.41 (s, 2H, C=CH-), 7.35 (d, 2H,
J = 6.4, benzene-H), 7.49 (d, 2H, J = 7.6, benzene-H). 13C NMR
(CDCl3, 100 MHz): 18.39, 19.24, 20.77, 27.44, 28.43, 28.64, 28.74,
30.24, 31.30, 31.78, 31.99, 36.66, 36.72, 37.66, 39.63, 40.21, 41.56,
Chem Biol Drug Des 2012; 79: 879–887
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