Synthesis and Properties of a New Multivalent Lipid
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5027
instruments). Samples were prepared from 10 mg/mL liposome
solutions and 5 mg/mL DNA solutions, using 100 µg of λ-phage
DNA (New England Biolabs) per sample. The CL-DNA
complexes were formed by combining the solutions, diluting
2-fold in DMEM, and incubating them at 4 °C for 2-3 days
following extensive centrifugation in an Eppendorf tube.
Samples were then transferred to 1.5 mm quartz capillaries
and flame-sealed. Typically the CL-DNA complexes formed
a white precipitate.
N1-(2-a m in oeth yl)-3,4-d i(oleyloxy)-ben za m id e (5). To
a solution of 4.00 g (6.11 mmol) of 4 in 10 mL of dichlo-
romethane, 0.77 g (6.72 mmol) of N-hydroxysuccinimide
(Bachem) and, after stirring for 5 min, 1.39 g (6.72 mmol) of
N,N′-dicyclohexylcarbodiimide (DCC, Sigma) in 2 mL of dichlo-
romethane were added. The reaction mixture was stirred for
2 h and then filtered. To the filtrate, a total of 4.1 mL (3.7 g,
61 mmol) of ethylenediamine was added quickly with stirring.
After being stirred for 3 h, the reaction mixture was filtered
and 200 mL of chloroform, 50 mL of methanol, and 100 mL of
water were added. The phases were separated after thorough
mixing. The organic phase was then extracted two more times
with water to which NaCl and some NaOH had been added,
dried (Na2SO4), and evaporated. The resulting residue was
purified by flash chromatography on 250 g of silica gel using
chloroform/methanol (9:1) as the eluent to yield 3.66 g (5.25
mmol, 86%) of 5 as an off-white, waxy solid. Rf ) 0.22 (9:1
CHCl3/MeOH). 1H NMR (CDCl3, 200 MHz): δ 0.84 (“t”, J )
6.7 Hz, 6 H, CH2-CH3), 1.0-2.5 (4 m, 56 H, alkyl-CH2), 2.90
(t, 3J ) 6.1 Hz, 2 H, H2N-CH2), 3.46 (q, 3J ) 5.9 Hz, 2 H,
C(O)-NH-CH2), 3.8-4.2 (m, 4 H, O-CH2), 5.2-5.5 (m, 4 H,
Syn th esis. Chemicals were from Fisher Scientific and at
least of analytical grade unless otherwise indicated.
NR,Nδ,Nδ-Tr is(2-cya n oeth yl)or n ith in e (1). To a solution
of 4.00 g (100 mmol) of sodium hydroxide in 120 mL of
methanol, a total of 16.9 g (100 mmol) of L-ornithine hydro-
chloride (Sigma) was added with stirring. After 2 min, a total
of 26.3 mL (21.2 g, 400 mmol) of acrylonitrile (Fluka) was
added in one portion. Stirring was continued for 24 h at room
temperature, and a total of 100 mL of methanol was added.
The reaction mixture was then acidified slightly by the
addition of concentrated hydrochloric acid (about 13 mL) and
was filtered immediately. The pH of the filtrate was adjusted
to 6 using 25% sodium hydroxide solution. After standing at
4 °C for 16 h, the mixture was filtered to yield 20.3 g (62 mmol,
62%) of 1 (hydrochloride form) as white crystals. Rf ) 0.79
(solvent mixture A). 1H NMR (D2O, 200 MHz): δ 1.5-2.3 (2
m, 4 H, CH2-CH2-CH), 2.6-3.4 (2 m, 12 H, CH2-CH2-CN),
3.4-4.0 (2 m, 3 H, CH2-CH2-CH2-CH). 13C NMR (D2O): δ
15.4, 15.8 (CH2-CN), 22.1 (CH2-CH2-CH), 27.7 (CH2-CH),
42.4, 48.6 (CH2-CH2-CN), 52.4 (CH2-CH2-CH2-CH), 63.0
(CH), 118.0, 121.2 (CN), 173.5 (CO2H).
3
dCH-), 6.61 (bt, 1 H, C(O)-NH), 6.81 (d, J ) 8.4 Hz, 1 H,
Har m-C(O)), 7.25 (dd, 3J ) 8.4 Hz, 4J ) 2.1 Hz, 1 H, Har p-OR),
7.38 (d, 4J ) 2.1 Hz, 1 H, Har o-C(O), o-OR). 13C NMR
(CDCl3): δ 14.5 (CH3), 23.1 (CH2-CH3), 26.5, 27.6, 29.7, 29.8,
30.0, 30.1, 30.2, 30.3, 32.3 (CH2-CH2-C), 40.6, 40.1 (CH2-
N), 69.8, 69.3 (CH2-O), 112.4, 113.3, 121.3, (aromatic CH),
125.9 (C-C(O)N), 130.2, 130.3 (dCH-CH2), 149.0, 152.6 (C-
O-CH2), 168.8 (C(O)NH).
N1-[2-((1S)-1-[ter t-Bu tylca r ba m oyla m in o(3-[ter t-bu tyl-
ca r b a m oyla m in o]p r op yl)a m in o]-4-[d i(3-[ter t-b u t ylca r -
ba m oyla m in o]p r op yl)a m in o]b u t ylca r boxa m id o)et h yl]-
3,4-d i[oleyloxy]ben za m id e (6). To a solution of 615 mg (0.88
mmol) of 5, 619 mg (0.88 mmol) of 3, and 135 mg (0.88 mmol)
of 1-hydroxybenzotriazole (HOBt, Peptides International) in
5 mL of dichloromethane, a total of 197 mg (0.955 mmol) of
DCC in 0.5 mL of dichloromethane was added. After being
stirred for 6 h, the reaction mixture was filtered and evapo-
rated. The resulting residue was dissolved in 40 mL of diethyl
ether, and the solution was washed with 10% citric acid, twice
with saturated NaHCO3, and with water. The ether solution
was dried (Na2SO4) and evaporated, and the residue was
purified by flash chromatography on 60 g of silica gel using
chloroform/methanol (15:1) as the eluent41 to yield 1.095 g
(0.792 mmol, 90%) of 6 as a colorless solid. Rf ) 0.32 (9:1
NR,Nδ,Nδ-Tr is(3-a m in op r op yl)or n ith in e (2). To a mix-
ture of 9.6 g (36 mmol) of 1 and 80 mL of 1.4 M sodium
hydroxide in 95% ethanol in a Parr hydrogenator flask, a total
of 5.00 g of a supension of Raney nickel in water (Fluka) was
added. The mixture was agitated for 16 h under 60 psi (4 bar)
of hydrogen pressure at room temperature. The Raney nickel
was removed by filtration, and the solvent was evaporated.
The resulting oil (15.5 g) was used for the next step without
further purification. Pure hydrochloride for analytical purposes
was obtained by adding concentrated hydrochloric acid to the
reaction solution and collecting and drying the precipitate. 1H
NMR (D2O, 200 MHz): δ 1.65-2.30 (m, 10 H, C-CH2-C),
2.95-3.50, (m, 14 H, CH2-N), 3.65-3.8 (m, 1 H, CH). 13C NMR
(D2O): δ 20.0, 22.2 (2 C), 24.3, 27.0 (C-CH2-C), 37.1 (3 C,
CH2-NH3+), 44.4 (CH2-NH2+), 50.4 (2 C), 52.6 (CH2-NH+),
62.3 (CH), 173.3 (CO2H).
1
CHCl3/MeOH). H NMR (D2O, 200 MHz): δ 0.85 (“t”, J ) 6.7
Hz, 6 H, CH2-CH3), 1.40 (s, 36 H, CH3), 1.0-2.6 (several b/m,
66 H, C-CH2-C), 2.8-3.3 (m, 14 H, CH2-N), 3.3-3.6 (b, 4
H, N-CH2-CH2-N), 3.8-4.2 (m, 4 H, O-CH2), 5.1-5.3 (b, 1
H, CH), 5.2-5.4 (m, 4 H, dCH-), 6.61 (bt, 1 H, C(O)-NH),
NR-ter t-Bu tylca r ba m oyl-NR,Nδ,Nδ-tr is(3-[ter t-bu tylca r -
ba m oyla m in o]p r op yl)or n ith in e (3). The raw product from
the previous step (15.5 g; assay calculated for 36 mmol ) 100%
yield from previous step) was dissolved in water/THF (1:1, v/v).
Over a period of 1 h, a solution of 34.5 g (158 mmol) of Boc2O
(Novabiochem) in 70 mL of THF and 40 mL of 4 M sodium
hydroxide in water were added in four portions with stirring.
The reaction mixture was kept at room temperature by cooling
with a water bath, and stirring was continued for 6 h. Most of
the THF was then evaporated. After addition of 250 mL of
diethyl ether, the mixture was acidified using half-concen-
trated hydrochloric acid. The phases were separated, and the
aqueous phase was extracted two more times with 150 mL of
ether. The combined organic phases were washed twice with
250 mL of water, dried (Na2SO4), and evaporated. The residue
was purified by flash chromatography on 800 g of silica gel
using chloroform/methanol (19:1) as the eluent41 to yield 22.5
g (32.0 mmol, 89%) of 3 as a colorless solid. Rf ) 0.40 (6:1
CHCl3/MeOH). 1H NMR (CDCl3, 200 MHz): δ 1.39 (s, 36 H,
CH3), 0.7-2.2 (2 m, 10 H, CH2-CH2-C), 2.8-3.4 (2 m, 14 H,
CH2-N), 4.0-4.2 (m, 1 H, CH), 5.0-5.6 (b, 2 H, NH), 5.9-6.3
(b, 1 H, NH). 13C NMR (CDCl3): δ 21.2 (-CH2-CH2-CH), 24.3
(-CH2-CH2-N-(CH2)3-CH), 27.4 (CH-N(Boc)-CH2-CH2),
28.8 (CCH3), 30.1 (-CH2-CH), 38.0 (CH2-N(H)Boc), 45.7
(CH2-N(Boc)-CH), 50.6, (CH2-N-(CH2)3-CH), 52.9 (CH2-
CH2-CH2-CH), 59.7 (CH), 79.9, 81.2 (CCH3), 156.9 (O-C(O)-
N), 173.9 (CO2H). MALDI-MS: m/z 705.8 (M + H+).
3
6.80 (d, J ) 8.3 Hz, 1 H, Har m-C(O)), 7.0-7.2 (b, NH), 7.33
(dd, 3J ) 8.3 Hz, 4J ) 2 Hz, 1 H, Har p-OR), 7.44 (d, 4J ) 2 Hz,
1 H, Har o-C(O), o-OR). 13C NMR (D2O): δ 14.5 (CH2-CH3),
23.1 (CH2-CH3), 26.5, 27.3, 27.6, 29.6, 29.7, 29.8, 29.9, 30.2
(CH2-CH2-CH2, CH-CH2), 32.3 (CH3-CH2-CH2), 28.7, 28.9
(C(CH3)3), 38.6, 39.5, 41.7 (CH2-N(H)-C(O)), 43.9 (CH2-
N(Boc)-CH), 52.0, 53.7, (CH2-N-CH2-(CH2)2-CH), 60.2
(CH), 69.5, 69.6 (CH2-O), 79.4, 81.3 (C(CH3)3), 112.6, 113.3,
120.3, (aromatic CH), 127.0 (Car-C(O)), 130.2, 130.4 (dCH-
CH2), 149.2, 152.2 (Car-O-CH2), 156.5 (O-C(O)-N), 167.8
(C(O)-Car), 173.3 (C(O)-CH). MALDI-MS: m/z 1382.6 (M +
H+).
N1-[2-((1S)-1-[(3-Am in op r op yl)a m in o]-4-[d i(3-a m in o-
p r op yl)a m in o]bu tylca r boxa m id o)eth yl]-3,4-d i[oleyloxy]-
ben za m id e (MVL5). To a concentrated solution of 1.00 g (0.72
mmol) of 6 in ethyl acetate, a total of 10 mL of a freshly
prepared saturated solution of HCl in ethyl acetate was added.
The mixture was stirred for 30 min at room temperature,
during which time a white precipitate formed, and evaporated.
To the residue, a total of 5 mL of ethyl acetate was added with
stirring and was evaporated. This was repeated to yield 830
mg (0.71 mmol, 99%) of MVL5 as a white powder. Rf ) 0.64
(2:2:1 CHCl3/MeOH/25% NH4OH). 1H NMR (CDCl3/MeOH-d4,
9:1, 200 MHz): δ 0.74 (“t”, J ) 6.7 Hz, 6 H, CH3), 0.9-1.5