Cationic Alkyl Acyl Carnitine Ester for Gene Delivery
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2213
University of California at San Francisco. TLC analyses of
lipids were performed on 0.25-mm silica gel plates. DMF was
distilled from CaH2, CH2Cl2 was distilled from P2O5, and THF
was distilled from Na metal and benzophenone. Column
chromatography was performed with silica gel (Aldrich, 230-
400 mesh). Unless noted otherwise, the ratios describing the
compositions of solvent mixtures represent relative volumes.
Elemental analyses were performed by Microanalytical Lab,
University of California at Berkeley. The lipids DOPE and
DOTAP were purchased from Avanti Polar Lipids, Alabaster,
AL. All other organic chemicals were purchased from Aldrich.
CV-1 cells (monkey fibroblast) were provided by the UCSF cell
culture facility. Female ICR mice were obtained from Simon-
sen (Gilroy, CA). All animals were handled in accordance with
protocols described by the National Institutes of Health Guide-
lines for the Care and Use of Laboratory Animals, and with
the approval of the Committee of Animal Research at the
University of California, San Francisco. Animals were sacri-
ficed at the stated times with a sodium pentabarbital overdose.
Plasmids pCMV-âGal and RSV-Luc were grown in Escherichia
coli, then extracted, and purified by two centrifugations in
equilibrium on CsCl gradients. The plasmid DNAs were
subjected to two CsCl gradients to ensure purity and had less
than 20 endotoxin units per milligram of DNA as measured
by the Whitaker Biotechnology endotoxin detection kit.
Gen er a l P r oced u r e for th e P r ep a r a tion of Alk yl Ca r -
n itin e Ester s. Exa m p les: Stea r yl Ca r n itin e Ester (2a ).
A solution of DL-carnitine hydrochloride (1.0 g, 5.05 mmol) and
sodium hydroxide (0.303 g, 7.58 mmol) in ethanol (15 mL) was
stirred at room temperature for 2 h. The resulting white
precipitate (NaCl) was removed by filtration, and the solvent
was evaporated under reduced pressure to give a white solid,
carnitine inner salt. A suspension of the carnitine inner salt
and 1-iodooctadecane (2.31 g, 6.06 mmol) in DMF-dioxane (3:
5, 40 mL) was heated with an oil bath at 120 °C under argon
for 4 h. The solvent was removed by a rotary evaporator
followed by high vacuum, and the residue was resolved by
chromatography on a silica gel column using CH3OH-CHCl3
as eluant to give 2.22 g (81%) of stearyl carnitine ester (2a )
as a white solid: 1H NMR (CDCl3) δ 4.79 (m, 1 H), 4.43 (d, J
) 5.3, 1 H), 4.09 (t, J ) 6.9, 2 H), 4.03 (d, J ) 13.0, 1 H), 3.67
(dd, J ) 10.3, 13.3, 1 H), 3.51 (s, 9 H), 2.79 (dd, J ) 5.7, 17.0,
1 H), 2.66 (dd, J ) 7.0, 17.1, 1 H), 1.80-1.60 (m, 4 H), 1.26
(brd s, 28 H), 0.88 (t, J ) 6.6, 3 H); LSIMS (NBA) m/e 414.4
for C25H52NO3 (cation).
Gen er a l P r oced u r e for th e P r ep a r a tion of Alk yl Acyl
Ca r n itin e Ester s. Exa m p le: Stea r yl Stea r oyl Ca r n itin e
Ester (Stea r yl 3-Stea r oyloxy-4-tr im eth yla m m on iu m Bu -
tyr a te Ch lor id e, 4a ). To a solution of stearic acid (2.0 g, 7.04
mmol) in CHCl3 (fresh dried, 30 mL) was added 1,1′-carbon-
yldiimidazole (0.736 g, 3.70 mmol), and the mixture was stirred
at room temperature under argon for 5 h. The formed white
precipitate (byproduct) was removed by filtration, and the
filtrate was evaporated under reduced pressure to give the
stearic anhydride as a white solid. IR showed an absorption
at 1804.5 cm-1. The anhydride was used for next step reaction
without further purification. A solution of the stearic anhy-
dride (1.94 g, 3.52 mmol), stearyl carnitine ester (0.953 g, 1.76
mmol), and 4-(dimethylamino)pyridine (0.429 g, 3.52 mmol)
in CHCl3 (dry, 15 mL) was stirred at room temperature under
argon for 4 days. The solvent was removed under reduced
pressure, and the residue was washed twice by cold diethyl
ether. The solid was chromatographed on a silica gel column
using MeOH-CHCl3 (1:5) as eluant to give the SSCE iodide.
The iodide was exchanged by chloride with an anion exchange
column to give 0.872 g (69%) of stearyl stearoyl carnitine ester
chloride (4a ) as a white solid: 1H NMR (300 MHz, CDCl3) δ
5.67 (q, 1 H), 4.32 (d, J ) 14.3, 1 H), 4.07 (m, 3 H), 3.51 (s, 9
H), 2.82 (t, J ) 5.9, 2 H), 2.33 (t, J ) 7.6, 2 H), 1.59 (brd m, 4
H), 1.25 (broad s, 58 H), 0.88 (t, J ) 6.6, 6 H); LSIMS (NBA)
m/e 680.6 for M+ (C43H86NO4). Anal. (C43H86ClNO4‚H2O) C,
H, N.
By this procedure were prepared the following alkyl acyl
carnitine esters.
L-Stea r yl stea r oyl ca r n itin e ester (L-stea r yl 3-stea r oyl-
oxy-4-tr im eth yla m m on iu m bu tyr a te ch lor id e, 4a ′) was
prepared with the same procedure using L-carnitine as starting
material. Analytical data are the same as for 4a .
Stea r yl oleoyl ca r n itin e ester (stea r yl 3-oleoyloxy-4-
tr im eth yla m m on iu m bu tyr a te ch lor id e, 4b): yield 41%;
1H NMR (300 MHz, CDCl3) δ 5.67 (m, 1 H), 5.35 (m, 2 H),
4.33 (d, J ) 14.5, 1 H), 4.08 (m, 3 H), 3.48 (s, 9 H), 2.83 (dd,
J ) 4.4, 6.8, 2 H), 2.34 (dd, J ) 6.3, 7.7, 2 H), 2.02 (brd m, 4
H), 1.26 (brd m, 54 H), 0.88 (t, J ) 6.7, 6 H); LSIMS (NBA)
m/e 678.7 for M+ (C43H84NO4). Anal. (C43H84ClNO4‚H2O) C,
H, N.
P a lm ityl p a lm itoyl ca r n itin e ester (p a lm ityl 3-p a lm i-
t oyloxy-4-t r im et h yla m m on iu m b u t yr a t e ch lor id e, 4c):
yield 65%; 1H NMR (300 MHz, CDCl3) δ 5.67 (q, 1 H), 4.33 (d,
J ) 14.0, 1 H), 4.07 (m, 3 H), 3.51 (s, 9 H), 2.82 (t, J ) 5.9, 2
H), 2.33 (t, J ) 7.6, 2 H), 1.59 (brd m, 4 H), 1.25 (brd s, 58 H),
By this procedure were prepared the following alkyl car-
nitine esters.
0.88 (t, J ) 6.52, 6 H); LSIMS (NBA) m/e 680.6 for M+ (C39H78
NO4). Anal. (C39H78ClNO4‚H2O) C, H, N.
-
P a lm ityl ca r n itin e ester (2b): yield 65%; 1H NMR
(CDCl3) δ 4.78 (m, 1 H), 4.44 (d, J ) 5.4, 1 H), 4.09 (t, J ) 6.9,
2 H), 3.65 (dd, J ) 10.2, 13.3, 1 H), 3.58 (d, J ) 5.1, 1 H), 3.51
(brd s, 9 H), 2.80 (dd, J ) 5.7, 17.2, 1 H), 2.66 (dd, J ) 7.1,
17.1, 1 H), 1.65 (broad m, 4 H), 1.26 (brd s, 24 H), 0.88 (t, J )
0.66, 3 H); LSIMS (NBA) m/e 386.2 for C23H48NO3 (cation).
Myr istyl m yr istoyl ca r n itin e ester (m yr istyl 3-m yr is-
t oyloxy-4-t r im et h yla m m on iu m bu t yr a t e ch lor id e, 4d ):
yield 35%; 1H NMR (300 MHz, CDCl3) δ 5.67 (q, 1 H), 4.32 (d,
J ) 13.8, 1 H), 4.07 (m, 3 H), 3.50 (s, 9 H), 2.82 (t, J ) 6.1, 2
H), 2.33 (t, J ) 7.6, 2 H), 1.61 (brd m, 4 H), 1.26 (brd s, 42 H),
0.88 (t, 6 H); LSIMS (NBA) m/e 568.6.7 for M+ (C35H70NO4).
Anal. (C35H70ClNO4‚1/2H2O) C, H, N.
L-Myr istyl m yr istoyl ca r n itin e ester (m yr istyl 3-m yr is-
toyloxy-4-tr im eth yla m m on iu m bu tyr a te ch lor id e, 4d ′)
was prepared with the same procedure using L-carnitine as
starting material. Analytical data are the same as for 4d , mp
157 °C dec.
Myr istyl ca r n itin e ester (2c): yield 74%; 1H NMR (CDCl3)
δ 4.79 (m, 1 H), 4.43 (d, J ) 5.3, 1 H), 4.09(t, J ) 6.9, 2 H),
4.03 (d, J ) 13.0, 1 H), 3.67 (dd, J ) 10.3, 13.3, 1 H), 3.51 (s,
9 H), 2.79 (dd, J ) 5.7, 17.0, 1 H), 2.66 (dd, J ) 7.0, 17.1, 1 H),
1.80-1.60 (m, 4 H), 1.26 (brd s, 20 H), 0.88 (t, J ) 6.6, 3 H);
LSIMS (NBA) m/e 358.1 for C21H44NO3 (cation).
La u r yl ca r n itin e ester (2d ): yield 77%; 1H NMR (CDCl3)
δ 4.80 (m, 1 H), 4.43 (d, J ) 5.3, 1 H), 4.09 (t, J ) 6.5, 2 H),
4.03 (d, J ) 10.9, 1 H), 3.67 (dd, J ) 10.2, 13.2, 1 H), 3.52 (s,
9 H), 2.79 (dd, J ) 5.7, 17.1, 1 H), 2.67 (dd, J ) 7.0, 17.1, 1 H),
1.63 (t, J ) 6.6, 2H), 1.26 (brd s, 18 H), 0.88 (t, J ) 6.6, 3 H);
LSIMS (NBA) m/e 330.3 for C19H40NO3 (cation).
La u r yl la u r oyl ca r n itin e ester (la u r yl 3-la u r oyloxy-4-
tr im eth yla m m on iu m bu tyr a te ch lor id e, 4e): yield 37%;
1H NMR (300 MHz, CDCl3) δ 5.64 (m, 1 H), 4.28 (d, J ) 14.0,
1 H), 4.07 (m, 3 H), 3.50 (s, 9 H), 2.82 (m, 2 H), 2.33 (t, J )
7.6, 2 H), 1.61 (brd m, 4 H), 1.26 (brd s, 38 H), 0.88 (t, 6 H);
LSIMS (NBA) m/e 512.46 for M+ (C31H62NO4). Anal. (C31H62
-
Oleyl Ca r n itin e Ester (2e). cis-1-Iodooctadec-9-ene was
prepared from oleyl alcohol with methyltriphenoxyphospho-
nium iodide according to Verheyden and Moffatt33 and used
to prepare 2e: yield 85%; 1H NMR (CDCl3) δ 5.33-5.45 (m, 2
H), 4.77 (m, 1 H), 4.05-4.20 (m, 3 H), 3.55-3.7 (m, 2 H), 3.50
(s, 9 H), 2.84 (dd, J ) 6.3, 17.1, 1 H), 2.66 (dd, J ) 8.2, 17.1,
1 H), 1.9-2.1 (brd m, 4 H), 1.55-1.7 (m, 2 H), 1.2-1.45 (m, 22
ClNO4‚21/2 H2O) C, H, N.
Oleyl oleoyl ca r n itin e ester (oleyl 3-oleoyloxy-4-tr i-
1
m eth yla m m on iu m bu tyr a te ch lor id e, 4f): yield 28%; H
NMR (300 MHz, CDCl3) δ 5.66 (m, 1 H), 5.35 (m, 4 H), 4.32
(d, J ) 14.6, 1 H), 4.09 (m, 3 H), 3.48 (s, 9 H), 2.82 (m, 2 H),
2.33 (t, J ) 7.6, 2 H), 2.00 (brd m, 8 H), 1.1-1.4 (brd m, 46 H),
H), 0.88 (t, J ) 6.6, 3 H); LSIMS (NBA) m/e 412.4 for C25H50
NO3 (cation).
-
0.88 (t, J ) 6.7, 6 H); LSIMS (NBA) m/e 676.6 for M+ (C43H82
NO4). Anal. (C43H82ClNO4‚2H2O) C, H, N.
-