Symmetrical cationic triglycerides: An efficient synthesis and application to gene transfer

Article abstract of DOI:10.1016/S0968-0896(00)00242-X

Some cationic triglycerides 1Aa-1Cb which have a symmetrical structure were effectively synthesized and formulated into cationic liposomes with the co-lipid dioleoylphosphatidylethanolamine (DOPE) and/or dilauroylphosphatidylcholine (DLPC). The plasmid encoding a luciferase was delivered into CHO cells by using these cationic liposomes. Our symmetrical cationic triglycerides showed high transfection activity when DOPE was used as a co-lipid. Among the symmetrical cationic triglycerides synthesized here, 1Ab and 1Ac, which have an oleoyl group at the 1- and 3-position in the glycerol backbone and also have a relatively long linker connecting the 2-hydroxy group in glycerol with the quaternary ammonium head group, were found to be the most suitable for gene delivery into cells. The transfection activity of the symmetrical cationic triglyceride 1Ab was comparable with that of its asymmetrical congener 6 and several times higher than that of Lipofectin.

Full text of DOI:10.1016/S0968-0896(00)00242-X

Bioorganic & Medicinal Chemistry 9 (2001) 245±254
Symmetrical Cationic Triglycerides:
An Ecient Synthesis and Application to Gene Transfer
Satoshi Obika, Wei Yu, Atsuko Shimoyama, Takeshi Uneda, Kazuyuki Miyashita,
Takefumi Doi and Takeshi Imanishi*
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
Received 13 June 2000; accepted 25 August 2000
AbstractÐSome cationic triglycerides 1Aa±1Cb which have a symmetrical structure were e€ectively synthesized and formulated
into cationic liposomes with the co-lipid dioleoylphosphatidylethanolamine (DOPE) and/or dilauroylphosphatidylcholine (DLPC).
The plasmid encoding a luciferase was delivered into CHO cells by using these cationic liposomes. Our symmetrical cationic tri-
glycerides showed high transfection activity when DOPE was used as a co-lipid. Among the symmetrical cationic triglycerides
synthesized here, 1Ab and 1Ac, which have an oleoyl group at the 1- and 3-position in the glycerol backbone and also have a
relatively long linker connecting the 2-hydroxy group in glycerol with the quaternary ammonium head group, were found to be the
most suitable for gene delivery into cells. The transfection activity of the symmetrical cationic triglyceride 1Ab was comparable with that
1
of its asymmetrical congener 6 and several times higher than that of Lipofectin . # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
been reported.5
À11
As shown in Fig. 1, the great major-
ity of the known cationic lipids for cationic liposome-
mediated gene transfer have an asymmetric
Genetic engineering, and molecular biology, gene
therapy has recently received a considerable amount of
attention owing to the rapid development of gene ana-
lytical techniques. One fundamental gene therapy tech-
nology is a gene delivery system. Among a large number
of gene delivery systems, such as microinjection, elec-
troporation, calcium phosphate precipitation, lipofec-
tion, and viral vector systems, the viral vector systems
were thought to be one of the most practical transfec-
4
,6À11
structure,
the same as natural phospholipids. The
asymmetric synthesis of the triglyceride was very
troublesome; therefore, many asymmetric cationic lipids
4
,6,7,11
were employed as a racemic mixture,
synthesized via the enzymatic hydrolysis of a relatively
or some were
7
expensive natural phospholipid. A simple strategy to
overcome these problems is to use a symmetrical catio-
nic triglyceride instead of an asymmetric one; however,
to our best knowledge, no application of cationic lipids
with a symmetric structure has been reported to date,
1
tion methods for in vivo applications. Although suc-
cessful viral vector-mediated gene transfers have been
reported, there are serious safety concerns about the
1
2À14
except for our previous results.
Here, we designed
2
clinical use of these systems.
and synthesized several symmetrical cationic triglycer-
ides 1 which were composed of biogenic materials, such
as fatty acids, glycerin, and amino acid derivatives, to
reduce the cytotoxicity of these cationic triglycerides.
The transfection eciency of the cationic liposomes
composed of the symmetrical triglycerides 1 and natural
phospholipids was also investigated.
On the other hand, one non-viral vector system, lipo-
fection, has been the focus of much recent research for
3
its safety, simple usage, and non-immunogenicity.
Since Felgner and co-workers reported the ®rst example
of ecient gene delivery into living cells with cationic
1
liposomes (Lipofectin ) composed of a synthetic catio-
nic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-
ammonium chloride (DOTMA), numerous examples
of such cationic liposome-mediated gene delivery have
4
Results and Discussion
Synthesis of the symmetrical cationic triglycerides
The synthetic route for symmetrical cationic triglycer-
ides 1Aa±1Cb is illustrated in Scheme 1. According to
*
8
Corresponding author. Tel.: +81-6-6879-8200; fax: +81-6-6879-
204; e-mail: imanishi@phs.osaka-u.ac.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00242-X

246
S. Obika et al. / Bioorg. Med. Chem. 9 (2001) 245±254
the literature,15,16 dihydroxyacetone was coupled with
fatty acids, such as oleic acid, elaidic acid and stearic
acid, to a€ord the corresponding diesters 2A±2C,
respectively. The centered carbonyl group of 2A±2C was
asymmetrical product was observed in the H and 13C
NMR spectra. The alcohols 3A±3C were treated with
chloroacetic anhydride or 4-chlorobutyryl chloride to
give the corresponding triglycerides 4Aa±4Cb in 76±
95% yields. Moreover, another triglyceride 4Ac was
obtained in 87% yield by the reaction of 3A with
5-chlorovaleryl chloride. Next, the chloroacetates 4Aa,
1
1
7
reduced with NaBH4 to give symmetrical alcohols 3A±
C in 76±85% yields. In spite of the possibility for an
3
acyl migration of the obtained alcohols 3A±3C, no
1
8
Figure 1. Structures of symmetrical cationic triglycerides 1 and other cationic lipids used in gene delivery vectors.
Scheme 1. (a) Ref. 14 for 2A; elaidic acid, DCC, DMAP, CH
B; NaBH , THF±benzene±H O (17:3:1), 84% for 3C; (c) chloroacetic anhydride, 2,6-lutidine, CH
ride, 2,6-lutidine, CH Cl or benzene, 81±92% for b series; 5-chlorovaleryl chloride, 2,6-lutidine, CH
6±94%; (e) sodium iodide, methyl ethyl keone, 88±95%; (f) trimethylamine, Et O, 57±78%.
2
Cl
2
, 72% for 2B; ref. 15 for 2C; (b) ref. 16 for 3A; NaBH
Cl , 76±95% for a series; 4-chlorobutyryl chlo-
Cl , 87% for 4Ac; (d) trimethylamine, Et O,
4 2
, THF±H O (15:1), 76% for
3
4
2
2
2
2
2
2
2
2
6
2

S. Obika et al. / Bioorg. Med. Chem. 9 (2001) 245±254
247
4
Ba and 4Ca were reacted with an excess amount of
It has been reported that the replacement of DOPE with
other natural lipids, which has no activity to form an
inverted hexagonal phase, will not attain enhanced
ꢀ
anhydrous trimethylamine at 0 C to a€ord the desired
symmetrical cationic triglycerides 1Aa, 1Ba and 1Ca,
respectively. On the other hand, the chlorobutyryl deri-
vatives 4Ab, 4Bb, 4Cb and chlorovaleryl derivative 4Ac
were converted to iodo derivatives 5Ab, 5Bb, 5Cb and
transfection ability similarly to that of DOPE.2
0À23
To
evaluate the e€ect of a co-lipid when it is employed with
symmetrical cationic lipids, one of our symmetric tri-
glycerides, 1Ab, was formulated into cationic liposomes
with a di€erent amount of DOPE and/or dilauroylphos-
5
Ac, respectively, in 88±95% yields by stirring in methyl
ethyl ketone with sodium iodide. Formation of ammo-
nium salts 1Ab, 1Bb, 1Cb and 1Ac was accomplished by
reaction of the corresponding iodo compounds 5Ab,
5
symmetrical structure of the obtained ammonium salts 1
2
4
phatidylcholine (DLPC). Their ability to deliver a
plasmid encoding a luciferase into CHO cells was stu-
died (Fig. 2). The cationic liposomes composed of the
triglyceride 1Ab alone demonstrated only moderate
transfection eciency; however, the addition of DOPE
greatly enhanced the transfection activity of symmetric
cationic triglyceride 1Ab. On the other hand, the lipo-
some formulations with DLPC lost their transfection
activity. All of these ®ndings are consistent with the known
properties of asymmetric cationic lipids previously repor-
ted. We thus suggested that DOPE is quite e€ective as a
co-lipid when it is used not only with an asymmetric
cationic lipid but also with a symmetric triglyceride. It is
noteworthy that the transfection activity of our cationic
Bb, 5Cb and 5Ac with anhydrous trimethylamine. The
1
3
is clearly substantiated by the C NMR spectrum
Table 1).
(
Transfection activity of the cationic triglycerides
Most of the reported asymmetric cationic lipids were
formulated into cationic liposomes with the co-lipid di-
oleoylphosphatidylethanolamine (DOPE),
is known to induce formation of an inverted hexagonal
4
,6À8,11
which
1
9
phase and promote destabilization of the membrane.
Table 1. Selected 13C NMR data (d ppm in CDCl
) for symmetrical cationic triglycerides 1Aa±1Cb
-CHˆCH-
3
Compound
-CH
2
-N+-(CH
3
)
3
2 3
-CH CH
1
1
1
1
1
1
1
Aa
Ab
Ac
Ba
Bb
Ca
Cb
173.51
173.39
173.39
173.46
173.30
173.49
173.40
164.33
171.23
171.88
164.31
171.12
164.33
171.19
130.01
130.01
130.01
130.44
130.39
Ð
129.67
129.67
129.65
130.10
130.06
Ð
61.26
61.60
61.64
61.19
61.55
61.21
61.57
63.09
65.59
66.51
63.06
65.52
63.07
65.54
54.34
53.84
53.80
54.23
53.75
54.29
53.77
14.09
14.11
14.09
14.07
14.02
14.09
14.09
Ð
Ð
5
Figure 2. E€ect of ratio of 1Ab, DOPE and DLPC on transfection eciency of cationic liposomes. CHO cells were inoculated at a density of 1Â10
1
ꢀ
cells per 35-mm plate. After incubation for 18 h at 37 C, the cells were washed twice with phosphate-bu€ered saline (PBS). Then, cationic lipo-
somes±plasmid DNA (pGVC) complexes (liposome 10 mM, DNA 0.5 mg/mL) in 2 mL OPTI-MEM medium were added to the cell culture plates.
After incubation for 24 h, the cells were harvested and luciferase activity was measured using luciferase assay system (PicaGene ).
TM

248
S. Obika et al. / Bioorg. Med. Chem. 9 (2001) 245±254
liposome, which is composed of symmetrical cationic
triglyceride 1Ab and DOPE (1Ab±DOPE=1:4), was
several times higher than that of the widely used Lipo-
structure was introduced into the hydrophobic chain of
2
1
the lipid. The decrease in bilayer sti€ness probably
2
1
enhances the fusion ability of cationic liposomes. That
seems to be the reason why cationic triglycerides 1Bb
and 1Ab show higher transfection activity than 1Cb. It
is interesting that low transfection activity was observed
when cationic triglycerides with a short linker (1Aa, 1Ba
and 1Ca) were used; on the contrary, high transfection
activity was observed when cationic triglycerides with a
relatively long linker (1Ab, 1Bb, 1Cb and 1Ac) were
used. In the case of cationic triglycerides with a short
linker, the hydrophilic quaternary ammonium group of
the cationic triglycerides would be embedded in the lipid
bilayer of liposomes. This would have prevented the
positive charge of the head group in cationic lipid from
interacting with the negative charge of DNA or cell
membrane. Therefore, cationic lipid with a relatively
long linker is suggested to be essential for high
transfection activity.
1
fectin (DOTMA±DOPE=1:1) in CHO cells.
To compare the transfection ability of a symmetrical
cationic triglyceride with that of an asymmetric trigly-
ceride, the symmetrical triglyceride 1Ab and its asym-
7
metric congener 6 were formulated into cationic
liposomes with DOPE (1Ab or 6±DOPE=1:4), respec-
tively. The transfection eciency of these liposomes was
evaluated, and it was found that the symmetrical trigly-
ceride 1Ab worked as well as the asymmetrical trigly-
ceride 6 (Fig. 3). This result clearly indicates that the
asymmetrical cationic triglycerides, the synthesis of
which is a troublesome procedure, could be replaced
with easily prepared symmetrical cationic triglycerides.
To study the relationship between the structure of
cationic triglyceride and transfection activity, the catio-
nic liposomes composed of triglycerides 1Aa±1Cb and
DOPE (1±DOPE=1:4) were prepared, and their trans-
fection eciency was evaluated (Table 2). These results
clearly show that the transfection eciency depends
upon both the structure of the acyl chain at the 1- and
the 3-position of glycerol backbone and the length of
the linker connecting the hydrophilic head group with
the glycerol backbone. The transfection activity was
increased when the unsaturated structure was intro-
duced in the middle of a long acyl chain (1Ab > 1Bb >
Conclusion
DOPE is essential for e€ective transfection when a
symmetrical cationic triglyceride is used, and an asym-
metrical structure of cationic lipids was not essential for
ecient transfection activity. The optimal structure of
our cationic triglyceride is the one with an unsaturated (Z-
ole®ne) acyl chain at the 1- and 3-positions and a relatively
long linker at the 2-position of the glycerol backbone.
1
Cb). The transfection activity of the cationic triglycer-
ide containing an oleoyl group (1Ab) was higher than
that of 1Bb which has an elaidoyl group. It was known
that the bilayer sti€ness decreased when an unsaturated
Experimental
General considerations
All melting points were measured on a Yanagimoto
1
micro melting point apparatus and are uncorrected. H
Table 2. Relationship between the structure of 1 and the transfection
eciency of the cationic liposomesa
Triglycerides
R
n
Relative luciferase
activity
1
(
/Lipofectin )
1
Aa
Ba
-(CH
2
2
)
7
7
CH=CH(CH
2
)
)
7
CH
3
3
1
1
0.0Æ0.0
(
Z)
CH=CH(CH
E)
1
-(CH
)
2
7
CH
0.0Æ0.0
(
1
1
Ca
Ab
-(CH
CH=CH(CH
Z)
CH=CH(CH
E)
16CH
2
)
16CH
3
1
3
0.0Æ0.0
-(CH
2
2
)
7
7
2
2
)
)
7
CH
3
3
10.4Æ0.7
(
1
1
1
Bb
Cb
Ac
-(CH
)
7
CH
3
3
4
3.2Æ0.4
1.7Æ0.5
8.0Æ0.9
(
-(CH
2
)
3
-(CH
2
)
7
CH=CH(CH
Z)
2
)
7
CH
3
(
Lipofectin1
1
Figure 3. Transfection eciency of cationic liposomes (1Ab±
DOPE=1:4) compared with that of cationic liposomes (6±
DOPE=1:4). See caption of Figure 2.
aSee captions in Figure 2.

S. Obika et al. / Bioorg. Med. Chem. 9 (2001) 245±254
249
and 1 C NMR spectra were recorded on a Varian VXR-
2
6
3
ꢀ
Mp 83±85 C (hexane±CHCl ) (lit.
3
16
84±86 C). 1H
ꢀ
1
1
13
00 ( H, 200 MHz), JEOL EX-270 ( H, 270 MHz; C,
1
NMR (CDCl ) d: 0.88 (6H, t, J=6.5 Hz, CH ), 1.18±
3
3
7.5 MHz) or JEOL GX-500 ( H, 500 MHz) spectro-
1.36 (56H, m, CH ), 1.62 (4H, m, OCOCH CH ), 2.42
2 2 2
meter. IR spectra were recorded on a JASCO FT/IR-
00 spectrometer. Mass spectra were measured on
(4H, t, J=7.5 Hz, OCOCH ), 4.74 (4H, s, CH CO-
2 2
CH2).
2
JEOL JMS-D300 or JMS-600 mass spectrometers.
Luciferase activity was measured on an EG&G Bert-
hold Lumat LB9501. For column chromatography,
Merck Kieselgel 60 (70±200 mesh) or Fuji Silysia BW-
1,3-Dioleoylglycerol (3A). According to the literature,17
compound 3A was prepared in the following manner.
To a stirred solution of compound 2A (1.00 g,
ꢀ
1
27ZH (100±200 mesh) was used. DOPE, DLPC and
1.62 mmol) in THF±H O (15:1, 16 mL) at 0 C was
2
the luciferase expression plasmid pGVC were purchased
from WAKO Pure Chemical Industries (Osaka, Japan).
added NaBH (100 mg, 2.59 mmol). The reaction mix-
4
ture was stirred for 20 min at the same temperature. The
solvent was concentrated, and the residue was dissolved
in Et O and washed with water. The organic layer was
1
1
Lipofectin and OPTI-MEM were purchased from
Gibco BRL (NY, USA). Luciferase assay system Pica-
2
TM
Gene
was obtained from Toyo Inki (Tokyo, Japan).
Asymmetrical cationic triglyceride 6 was prepared
dried (MgSO ) and concentrated. The residue was
4
ꢀ
washed with n-pentane at À78 C to give a colorless oil
7
according to the literature.
3A (0.86 g, 85%).
1
H NMR (CDCl ) d: 0.88 (6H, t, J=7.0 Hz, CH ), 1.18±
3
3
1
.46 (40H, m, CH ), 1.64 (4H, m, OCOCH CH ), 2.01
2 2 2
Chemistry
(
8H, m, CH CHˆCHCH ), 2.34 (4H, t, J=7.5 Hz,
2
2
OCOCH ), 4.14±4.16 (5H, m, CH CHCH ), 5.34 (4H,
2 2 2
1
3
1,3-Dioleoyloxypropan-2-one (2A). Compound 2A was
5
prepared according to the literature.
m, CH CHˆCHCH ). C NMR (CDCl ) d: 174.33,
2
2
3
1
130.44, 130.14, 68.79, 65.46, 34.52, 33.03, 32.34, 30.11,
9.95, 29.89, 29.76, 29.68, 29.53, 29.41, 27.63, 27.59,
25.30, 23.11, 14.55.
2
ꢀ
15
ꢀ
1
Mp 40±41.5 C (MeOH) (lit. 43 C). H NMR (CDCl )
3
d: 0.88 (6H, t, J=6.5 Hz, CH ), 1.14±1.44 (40H, m, CH ),
3
2
1
.67 (4H, m, OCOCH CH ), 2.02, 2.17 (4H each, m,
1,3-Dielaidoylglycerol (3B). To a stirred solution of
compound 2B (1.00 g, 1.62 mmol) in THF±H O (15:1,
16 mL) at 0 C was added NaBH (100 mg, 2.59 mmol).
4
2
2
CH CHˆCHCH ), 2.42 (4H, t, J=7.5 Hz, OCOCH ),
2
2
2
2
ꢀ
4.75
(4H,
s,
CH COCH ),
2
5.34
(4H,
m,
2
CH CHˆCHCH ).
The reaction mixture was stirred for 20 min at the same
temperature. The solvent was concentrated, and the
residue was dissolved in Et O and washed with water.
2
2
1
,3-Dielaidoyloxypropan-2-one (2B). To a stirred solu-
2
tion of 1,3-dihydroxyacetone (0.82 g, 8.87 mmol) in
CH Cl (18 mL) were added elaidic acid (5.04 g,
The organic layer was dried (MgSO ) and concentrated.
4
ꢀ
The residue was washed with n-pentane at À78 C to
2
2
1
1
7.7 mmol) and 4-dimethylaminopyridine (2.16 g,
7.7 mmol). To this reaction mixture a solution of 1,1-
give a colorless oil 3B (0.76 g, 76%).
1
dicyclohexylcarbodiimide (3.70 g, 18.0 mmol) in CH Cl2
2
H NMR (CDCl ) d: 0.88 (6H, t, J=6.0 Hz, CH ), 1.15±
3
3
(
7 mL) was added dropwise with stirring at room tem-
1.40 (40H, m, CH ), 1.63 (4H, m, OCOCH CH ), 2.00
(8H, m, CH CHˆCHCH ), 2.35 (4H, t, J=7.0 Hz,
2 2 2
2 2 2
perature. After stirring for 20 h at the same temperature,
the precipitated cyclohexylurea was removed by ®ltra-
tion. The ®ltrate was washed with 5% HCl, H O, and
sat. NaCl. The organic layer was dried (Na SO ) and
2
concentrated, and the residue was recrystallized from
AcOEt±EtOH to give colorless crystals 2B (3.96 g,
2
2
OCOCH ), 4.10±4.20 (5H, m, CH CHCH ), 5.38 (4H,
1
3
m, CH CHˆCHCH ). C NMR (CDCl ) d: 173.87,
2
2
2
3
130.48, 130.17, 68.39, 65.03, 34.07, 32.58, 32.52, 31.88,
29.63, 29.54, 29.47, 29.29, 29.17, 29.08, 28.92, 24.87,
22.66, 14.09.
4
7
2%).
1,3-Distearoylglycerol (3C). To a stirred solution of
compound 2C (0.12 g, 0.1 mmol) in THF±benzene±H O
(17:3:1, 21 mL) at 0 C was added NaBH (12.5 mg,
4
ꢀ
731, 1469, 1420, 1301, 1251, 1210 cm
Mp 57.5±59 C (AcOEt±EtOH). IR ꢀ (KBr): 2918, 2850,
2
À1
1
ꢀ
1
.
H NMR
(
(
(
CDCl ) d: 0.88 (6H, t, J=6.5 Hz, CH ), 1.06±1.44
0.33 mmol). The reaction mixture was stirred for 1 h at
the same temperature. The solvent was concentrated,
and the residue was dissolved in chloroform and washed
3
3
40H, m, CH ), 1.62 (4H, m, OCOCH CH ), 1.95, 1.97
4H each, m, CH CHˆCHCH ), 2.42 (4H, t, J=7.5 Hz,
2
2
2
2
2
OCOCH ), 4.75 (4H, s, CH COCH ), 5.38 (4H, m,
2
with water. The organic layer was dried (MgSO ) and
4
2
2
1
3
CH CHˆCHCH ).
C NMR (CDCl ) d: 198.10,
concentrated. The residue was recrystallized from
CHCl to give colorless crystals 3C (0.10 g, 84%).
2
2
3
1
3
2
72.90, 130.48, 130.17, 130.20, 96.12, 66.13, 33.72,
2.61, 32.55, 31.90, 29.65, 29.55, 29.49, 29.40, 29.32,
9.18, 29.09, 29.03, 28.92, 24.78, 22.69, 14.12. MS (EI)
+
3
ꢀ
Mp 75±77 C (CHCl ) (lit.
3
16
76±78 C). 1H NMR
ꢀ
m/z: 618 (M , 2.5), 265 (35.7), 55 (100). Anal. calcd for
C H O : C, 75.67; H, 11.40. Found: C, 75.56; H,
1
(CDCl ) d: 0.87 (6H, t, J=6.0 Hz, CH ), 1.28±1.38
3
3
(56H, m, CH ), 1.62 (4H, m, OCOCH CH ), 2.32 (4H,
2 2 2
3
9
70
5
1
3
1.28.
t, OCOCH ), 4.06±4.22 (5H, m, CH CHCH ).
2
C
2
2
NMR (CDCl ) d: 173.90, 68.34, 65.00, 34.08, 31.93,
29.86, 29.70, 29.61, 29.53, 29.46, 29.36, 29.26, 29.12,
24.87, 24.80, 22.69, 14.14.
3
1
prepared according to the literature.
,3-Distearoyloxypropan-2-one (2C). Compound 2C was
1
6

250
S. Obika et al. / Bioorg. Med. Chem. 9 (2001) 245±254
1,3-Dioleoyl-2-chloroacetylglycerol (4Aa). To a stirred
solution of compound 3A (1.00 g, 1.61 mmol) in CH Cl
OCOCH CH ), 1.82 (2H, m, CH CH (CH ) Cl), 2.00,
2 2 2 2 2 2
2.02 (4H each, m, CH CHˆCHCH ), 2.32 (4H, t,
2
2
2
2
(
(
5 mL) at room temperature were added 2,6-lutidine
5 mL) and chloroacetic anhydride (0.80 g, 4.68 mmol).
J=7.5 Hz, OCOCH ), 2.35 (2H, t, J=6.5 Hz, OCO-
2
CH (CH ) Cl), 3.18 (2H, t, J=6.0 Hz, (CH ) CH Cl),
2
2 3
2 3
2
The reaction mixture was stirred for 3 h, and the solvent
was concentrated. The residue was dissolved in AcOEt,
washed with H O, sat. NaHCO , H O, 2% HCl, H O,
4.14, 4.32 (4H, ABX, J=6.0, 4.0 Hz, CH CHCH ), 5.27
2 2
(1H, m, CH CHCH ), 5.34 (4H, m, CH CHˆCHCH ).
2
2
2
2
1
3
C NMR (CDCl ) d: 173.24, 172.18, 130.01, 129.70,
2
3
2
2
3
and sat. NaCl. The organic layer was dried (MgSO4)
and concentrated, and the residue was puri®ed by ¯ash
column chromatography (hexane±AcOEt, 6:1) to a€ord
a colorless oil 4Aa (0.86 g, 77%).
69.22, 62.03, 44.30, 34.02, 33.28, 31.90, 31.72, 29.76,
29.71, 29.53, 29.31, 29.17, 29.11, 29.08, 27.23, 27.17,
24.84, 22.68, 22.16, 14.11. MS (EI) m/z: 738 (M , 3.3),
602 (14.9), 457 (80.2), 339 (10.6), 265 (39.9), 247 (10.9),
+
195 (9.5), 193 (27.6), 121 (22.0), 119 (49.7), 55 (100).
Anal. calcd for C H O Cl: C, 71.46; H, 10.77; Cl,
4.79. Found: C, 71.39; H, 10.64; Cl, 4.90.
IR ꢀ (KBr): 2924, 2855, 2032, 1748, 1653, 1457,
4
4
79
6
À1
1
1
CH3), 1.00±1.40 (40H, m, CH2), 1.60 (4H, m,
158 cm . H NMR (CDCl ) d: 0.88 (6H, t, J=6.0 Hz,
3
OCOCH CH ), 2.00, 2.02 (4H each, m,
2
1,3-Dielaidoyl-2-chloroacetylglycerol (4Ba). Compound
4Ba was prepared as described for 4Aa using 3B (0.47 g,
0.75 mmol) and chloroacetic anhydride (0.39 g,
2.28 mmol). The residue was puri®ed by ¯ash column
chromatography (hexane±AcOEt, 20:1) to a€ord a
colorless oil 4Ba (0.40 g, 76%).
2
CH CHˆCHCH ), 2.32 (4H, t, J=7.5 Hz, OCOCH ),
2
2
2
4
3
.07 (2H, s, CH Cl), 4.17, 4.36 (4H, ABX, J=6.0,
2
.5 Hz, CH CHCH ), 5.31 (1H, m, CH CHCH ), 5.34
2
2
2
2
1
3
(
4H, m, CH CHˆCHCH ). C NMR (CDCl ) d:
2
2
3
1
4
2
73.63, 167.04, 130.44, 130.26, 130.14, 71.70, 62.16,
1.08, 34.41, 32.35, 30.53, 30.45, 30.21, 30.13, 29.97,
9.77, 29.53, 27.90, 27.66, 27.62, 25.25, 23.13, 14.56. MS
IR ꢀ (KBr): 2924, 2854, 1744, 1465, 1285, 1242, 1163,
+
À1
1
(
EI) m/z: 696 (M , 8.8), 417 (17.2), 415 (38.6), 339 (5.4),
1092 cm . H NMR (CDCl ) d: 0.88 (6H, t, J=6.5 Hz,
3
2
7
4
65 (80.3), 55 (100). Anal. calcd for C H O Cl: C,
4
0.60; H, 10.55; Cl, 5.08. Found: C, 70.79; H, 10.48; Cl,
.87.
CH ), 1.10±1.45 (40H, m, CH ), 1.60 (4H, m, OCO-
CH CH ), 1.95, 1.97 (4H each, m, CH CHˆCHCH ),
1
73
6
3
2
2
2
2
2
2.32 (4H, t, J=7.5 Hz, OCOCH ), 4.08 (2H, s, CH Cl),
2 2
4.17, 4.36 (4H, ABX, J=6.0, 4.0 Hz, CH CHCH ), 5.34
2 2
1
,3-Dioleoyl-2-(4-chlorobutanoyl)glycerol (4Ab). Com-
(1H, m, CH CHCH ), 5.38 (4H, m, CH CHˆCHCH ).
2
2
2
2
1
3
pound 4Ab was prepared as described for 4Aa using 3A
2.33 g, 3.75 mmol) and 4-chlorobutyryl chloride (1.59 g,
1.3 mmol). The residue was puri®ed by ¯ash column
C NMR (CDCl ) d: 173.30, 173.26, 166.63, 71.27,
3
(
1
61.74, 40.66, 33.97, 32.62, 62.56, 32.38, 31.91, 29.67,
29.58, 29.50, 29.33, 29.20, 29.11, 29.06, 28.95, 28.53,
+
chromatography (hexane±AcOEt, 30:1) to a€ord a
colorless oil 4Ab (2.20 g, 81%).
24.81, 24.68, 22.69, 14.14. MS (EI) m/z: 696 (M , 10.8),
417 (20.1), 415 (44.0), 265 (86.8), 264 (100). Anal. calcd
for C H O Cl: C, 70.60; H, 10.55; Cl, 5.08. Found: C,
70.62; H, 10.30; Cl, 5.08.
4
1
73
6
IR ꢀ (KBr): 2930, 2854, 2676, 2031, 1746, 1652, 1456,
1377, 1166, 1049 cm . H NMR(CDCl ) d: 0.88 (6H,
t, J=6.5 Hz, CH , 1.18±1.42 (40H, m, CH ), 1.61 (4H,
À1
1
3
3
)
2
m, OCOCH CH ), 2.02, 2.00 (4H each, m,
2
1,3-Dielaidoyl-2-(4-chlorobutanoyl)glycerol (4Bb). Com-
pound 4Bb was prepared as described for 4Aa using 3B
(0.76 g, 1.22 mmol) and 4-chlorobutyryl chloride (0.68 g,
3.66 mmol). The residue was puri®ed by ¯ash column
chromatography (hexane±AcOEt, 20:1) to a€ord a
colorless oil 4Bb (0.76 g, 86%).
2
CH CHˆCHCH ), 2.10 (2H, m, COCH CH CH Cl),
2
2
2
2
2
2
J=7.0 Hz, COCH CH CH Cl), 3.60 (2H, t, J=6.5 Hz,
.32 (4H, t, J=7.5 Hz, OCOCH ), 2.53 (2H, t,
2
2
2
2
COCH CH CH Cl), 4.14, 4.33 (4H, ABX, J=6.0,
2
2
2
4
.0 Hz, CH CHCH ), 5.27 (1H, m, CH CHCH ), 5.34
2 2 2 2
1
3
(
4H, m, CH CHˆCHCH ). C NMR (CDCl ) d:
2
2
3
1
3
2
73.23, 171.73, 129.99, 129.69, 69.36, 61.96, 43.79,
3.98, 31.88, 31.09, 29.74, 29.67, 29.51, 29.29, 29.13,
9.06, 27.50, 27.19, 27.14, 24.82, 22.66, 14.09. MS (EI)
IR ꢀ (KBr): 2924, 2853, 2675, 2025, 1746, 1465, 1377,
À1
1
1167, 1096 cm
J=7.0 Hz, CH , 1.16±1.40 (40H, m, CH ), 1.61 (4H, m,
. H NMR (CDCl ) d: 0.88 (6H, t,
3
3
)
2
+
m/z: 724 (M , 6.1), 602 (14.2), 445 (40.1), 443 (100), 339
OCOCH CH ), 1.95, 1.97 (4H each, m, CH CHˆCH-
2
2
2
(
(
12.8), 265 (58.5), 181 (14.3), 179 (43.1), 107 (31.1), 105
84.3). Anal. calcd for C H O Cl: C, 71.18; H, 10.70;
CH ), 2.10 (2H, m, COCH CH CH Cl), 2.31 (4H, t,
2 2 2 2
J=7.5 Hz, OCOCH ), 2.53 (2H, t, J=7.0 Hz, COCH -
2
4
3
77
6
2
Cl, 4.89. Found: C, 71.19; H, 10.65; Cl, 4.83.
CH CH Cl), 3.60 (2H, t, J=6.5 Hz, COCH CH -
2 2 2 2
CH Cl), 4.14, 4.32 (4H, ABX, J=6.0, 4.0 Hz,
2
1
,3-Dioleoyl-2-(5-chlorovaleryl)glycerol (4Ac). Com-
pound 4Ac was prepared as described for 4Aa using 3A
0.38 g, 0.61 mmol) and 5-chlorovaleryl chloride (0.29 g,
.87 mmol). The residue was puri®ed by ¯ash column
CH CHCH ), 5.27 (1H, m, CH CHCH ), 5.38 (4H, m,
2
2
2
2
1
3
CH CHˆCHCH ).
C NMR (CDCl ) d: 173.26,
2
2
3
(
1
171.73, 130.48, 130.15, 69.36, 61.96, 43.81, 34.06, 32.58,
32.54, 31.88, 31.11, 29.74, 29.63, 29.56, 29.47, 29.29,
29.17, 29.09, 29.06, 28.93, 27.50, 24.82, 22.66, 14.09. MS
chromatography (hexane±AcOEt, 30:1) to a€ord a
colorless oil 4Ac (0.40 g, 87%).
+
(EI) m/z: 724 (M , 4.8), 706 (1.6), 602 (13.6), 445 (38.2),
443 (94.7), 339 (12.6), 265 (29.8), 181 (14.4), 179 (43.5),
IR ꢀ (KBr): 2922, 2855, 2677, 2030, 1745, 1655, 1459,
107 (32.9), 105 (91.4), 55 (100). Anal. calcd for
C H O Cl: C, 71.19; H, 10.70; Cl, 4.89. Found: C,
71.10; H, 10.43; Cl, 4.88.
À1
1
1
J=7.0 Hz, CH ), 1.20±1.42 (42H, m, CH ), 1.61 (4H, m,
377, 1162 cm
. H NMR (CDCl ) d: 0.88 (6H, t,
3
43 77
6
3
2

S. Obika et al. / Bioorg. Med. Chem. 9 (2001) 245±254
251
1,3-Distearoyl-2-chloroacetylglycerol (4Ca). To a stirred
solution of compound 3C (1.70 g, 2.72 mmol) in CH Cl
chromatography (hexane±AcOEt, 30:1) to a€ord a
colorless oil 5Ab (2.18 g, 88%).
2
2
(
(
16 mL) at room temperature were added 2,6-lutidine
16 mL) and chloroacetic anhydride (1.40 g, 8.19 mmol).
IR ꢀ (KBr): 2926, 2854, 1745, 1656, 1462, 1377,
À1
1
The reaction mixture was stirred for 3 h, and the solvent
was concentrated. The residue was dissolved in CHCl3,
washed with H O, sat. NaHCO , H O, 2% HCl, H O,
1167 cm . H NMR (CDCl ) d: 0.88 (6H, t, J=6.5 Hz,
3
CH3), 1.17±1.41 (40H, m, CH2), 1.62 (4H, m,
OCOCH CH ),
2.00,
2.02
(4H
each,
m,
2
3
2
2
2
2
and sat. NaCl. The organic layer was dried (MgSO4)
and concentrated, and the residue was puri®ed by SiO2
column chromatography (CH Cl ±AcOEt, 25:1) to
CH CHˆCHCH ), 2.13 (2H, m, COCH CH CH I),
2
2
2
2
2
2.32 (4H, t, J=7.5 Hz, OCOCH ), 2.48 (2H, t,
2
J=7.0 Hz, COCH CH CH I), 3.24 (2H, t, J=7.0 Hz,
2
2
2
2
2
a€ord a white powder 4Ca (1.81 g, 95%).
COCH CH CH I), 4.16, 4.32 (4H, ABX, J=6.0,
2 2 2
4
.5 Hz, CH CHCH ), 5.27 (1H, m, CH CHCH ), 5.34
2 2 2 2
13
ꢀ
Mp 58±60 C. IR ꢀ (KBr): 2915, 2849, 1739, 1467, 1416,
(4H, m, CH CHˆCHCH ). C NMR (CDCl ) d:
2
2
3
À1
1
1254, 1168, 1105 cm . H NMR (CDCl ) d: 0.88 (6H, t,
J=6.5 Hz, CH ), 1.10±1.40 (56H, m, CH ), 1.60 (4H, m,
OCOCH CH ), 2.32 (4H, t, J=7.0 Hz, OCOCH ), 4.08
2
(2H, s, CH Cl), 4.17, 4.36 (4H, ABX, J=6.0, 4.0 Hz,
1
CH CHCH ), 5.33 (1H, m, CH CHCH ). C NMR
CDCl ) d: 173.24, 166.60, 96.10, 77.47, 77.20, 77.00,
6.51, 71.25, 61.71, 40.63, 33.96, 31.92, 30.21, 29.67,
9.60, 29.44, 29.35, 29.22, 29.08, 24.80, 22.68, 22.68,
173.23, 171.41, 130.01, 129.69, 69.40, 69.18, 34.70,
34.02, 31.88, 30.30, 29.74, 29.69, 29.51, 29.31, 29.15,
29.09, 28.32, 27.21, 27.15, 24.82, 22.66, 14.09, 4.94. MS
3
3
2
2
2
+
(EI) m/z: 816 (M , 7.5), 798 (1.7), 602 (17.5), 535 (100),
2
3
339 (14.7), 271 (30.1), 265 (51.9), 197 (47.4). Anal. calcd
for C H O I: C, 63.21; H, 9.50; I, 15.53. Found: C,
63.27; H, 9.41; I, 15.31.
2
2
2
2
(
3
43 77
6
7
2
1
+
4.09. MS (EI) m/z: 700 (M , 2.7), 419 (15.9), 417
43.1), 341 (11.3), 267 (61.8), 57 (100). Anal. calcd for
C H O Cl: C, 70.20; H, 11.06; Cl, 5.05. Found: C,
1,3-Dioleoyl-2-(5-iodovaleryl)glycerol (5Ac). Compound
5Ac was prepared as described for 5Ab using 4Ac
(0.31 g, 0.42 mmol) and sodium iodide (0.70 g,
4.67 mmol). The residue was puri®ed by ¯ash column
chromatography (hexane±AcOEt, 100:1) to a€ord a
colorless oil 5Ac (0.33 g, 95%).
(
4
1
77
6
7
0.10; H, 10.79; Cl, 4.85.
1
,3-Distearoyl-2-(4-chlorobutanoyl)glycerol (4Cb). To a
stirred solution of compound 3C (0.77 g, 1.23 mmol) in
benzene (10 mL) at room temperature were added 2,6-
lutidine (10 mL) and 4-chlorobutyryl chloride (0.53 g,
IR ꢀ (KBr): 2926, 2854, 2026, 1748, 1653, 1456, 1373,
À1
1
1161 cm . H NMR (CDCl ) d: 0.88 (6H, t, J=7.0 Hz,
3
3
.75mmol). The reaction mixture was stirred for 3 h, and
CH , 1.19±1.41 (40H, m, CH2), 1.61 (4H, m,
OCOCH CH ), 1.75 (2H, m, CH CH (CH ) I), 1.85
2 2 2 2 2 2
3
)
the solvent was concentrated. The residue was dissolved in
CHCl , washed with H O, sat. NaHCO , H O, 2% HCl,
H O, and sat. NaCl. The organic layer was dried
(MgSO ) and concentrated, and the residue was recrys-
tallized from AcOEt to a€ord colorless crystals 4Cb
(2H, m, CH CH (CH ) I), 2.00, 2.02 (4H each, m,
2
3
2
3
2
2
2 2
CH CHˆCHCH ), 2.32 (4H, t, J=7.5 Hz, OCOCH ),
2
2
2
2
2.36 (2H, t, J=7.0 Hz, COCH CH CH I), 3.18 (2H, t,
2 2 2
4
J=7.0 Hz, COCH CH CH I), 4.14, 4.32 (4H, ABX,
2 2 2
(
0.83 g, 92%).
J=6.0, 4.0 Hz, CH CHCH ), 5.26 (1H, m,
2 2
1
3
CH CHCH ), 5.34 (4H, m, CH CHˆCHCH ).
C
2
2
2
2
ꢀ
471, 1255, 1175, 1108, 1066, 1041 cm
Mp 56±57 C (AcOEt). IR ꢀ (KBr): 2914, 2848, 1733,
1
(
(
m, COCH CH CH Cl), 2.32 (4H, t, J=7.5 Hz,
2
OCOCH ), 2.53 (2H, t, J=7.0 Hz, COCH CH CH Cl),
2
NMR (CDCl ) d: 173.23, 172.11, 130.01, 129.69, 62.22,
3
À1
1
.
H NMR
62.04, 34.02, 32.92, 32.56, 31.88, 29.74, 29.69, 29.51,
29.29, 29.15, 29.04, 27.21, 27.15, 25.66, 24.82, 22.66,
CDCl ) d: 0.88 (6H, t, J=6.5 Hz, CH , 1.18±1.38
3
3)
+
56H, m, CH ), 1.64 (4H, m, OCOCH CH ), 2.10 (2H,
14.09, 5.48. MS (EI) m/z: 830 (M , 4.8), 602 (13.3), 549
(69.2), 339 (13.8), 285 (19.6), 265 (29.8), 211 (20.7), 55
2
2
2
2
2
ꢁ
(100). Anal. calcd for C H O I 1/10 hexane: C, 63.80;
44 79
2
2
2
6
3
.60 (2H, t, J=6.0 Hz, COCH CH CH Cl), 4.14, 4.32
2
H, 9.65; I, 15.11. Found: C, 63.99; H, 9.53; I, 14.96.
2
2
(
4H, ABX, J=6.0, 4.0 Hz, CH CHCH ), 5.27 (1H, m,
2 2
1
3
CH CHCH ). C NMR (CDCl ) d: 173.28, 171.75,
1,3-Dielaidoyl-2-(4-iodobutanoyl)glycerol (5Bb). Com-
pound 5Bb was prepared as described for 5Ab using 4Bb
(0.76 g, 1.05 mmol) and sodium iodide (1.59 g,
10.6 mmol). The residue was puri®ed by ¯ash column
chromatography (hexane±AcOEt, 40:1) to a€ord a
colorless oil 5Bb (0.79 g, 92%).
2
2
3
9
6.12, 69.40, 61.96, 43.81, 34.04, 31.92, 31.13, 29.69,
.62, 29.47, 29.36, 29.27, 29.11, 27.51, 24.85, 22.68, 14.11.
9
MS (EI) m/z: 728 (M , 1.1), 606 (13.1), 445 (92.4), 341
+
(21.3), 267 (72.1), 181 (19.3), 179 (57.2), 107 (31.2), 105
(92.4), 57 (100). Anal. calcd for C H O Cl: C, 70.79; H,
81 6
43
11.19; Cl, 4.85. Found: C, 70.50; H, 10.97; Cl, 4.59.
IR ꢀ (KBr): 2922, 2853, 1744, 1457, 1375, 1165 cm . 1H
À1
1,3-Dioleoyl-2-(4-iodobutanoyl)glycerol (5Ab). To a stir-
red solution of sodium iodide (4.40 g, 29.7 mmol) in
NMR (CDCl ) d: 0.88 (6H, t, J=6.5 Hz, CH , 1.17±
1.42 (40H, m, CH ), 1.61 (4H, m, OCOCH CH ), 1.95,
2 2 2
3
3)
anhydrous methyl ethyl ketone (32 mL) was added 4Ab
(
1.97 (4H each, m, CH CHˆCHCH ), 2.13 (2H, m,
2
2
2.20 g, 3.03 mmol). The stirring was continued for 48 h
COCH CH CH I), 2.32 (4H, t, J=7.5 Hz, OCOCH ),
2 2 2 2
at room temperature and the reaction mixture was con-
centrated. The residue was dissolved in AcOEt, washed
with H O, 10% NaHSO , H O, 5% NaHCO , H O and
sat. NaCl. The organic layer was dried (MgSO ) and
4
concentrated. The residue was puri®ed by ¯ash column
2.47 (2H, t, J=7.0 Hz, COCH CH CH I), 3.24 (2H, t,
2 2 2
J=6.5 Hz, COCH CH CH I), 4.14, 4.32 (4H, ABX,
2
2
2
J=6.0, 4.0 Hz, CH CHCH ), 5.26 (1H, m,
2
2
3
2
3
2
2
1
3
CH CHCH ), 5.37 (4H, m, CH CHˆCHCH ).
C
2
2
2
2
NMR (CDCl ) d: 173.24, 171.41, 130.48, 130.15, 69.38,
3

252
S. Obika et al. / Bioorg. Med. Chem. 9 (2001) 245±254
6
2
2
7
2
1.96, 34.68, 34.00, 32.58, 32.54, 32.28, 31.88, 29.63,
9.56, 29.47, 29.29, 29.17, 29.09, 29.06, 28.95, 28.30,
4.82, 22.66, 14.11, 4.98. MS (EI) m/z: 816 (M , 8.1),
98 (1.8), 602 (13.6), 535 (80.5), 339 (14.9), 265 (44.7),
1Ab was prepared as described for 1Aa using 5Ab
(1.58 g, 1.94 mmol). The residue was puri®ed by SiO2
column chromatography (CHCl ±MeOH, 20:1) to
3
+
a€ord a white powder 1Ab (1.32 g, 78%).
71 (30.5), 197(49.4), 55 (100). Anal. calcd for
ꢁ
ꢀ
C H O I 1/10 hexane: C, 63.74, H, 9.68; I, 15.13.
4
Found: C, 63.59; H, 9.38; I, 14.84.
Mp 47±50 C. IR ꢀ (KBr): 2924, 2852, 1738, 1466, 1412,
1364, 1252, 1176, 1117, 1067, 1025, 968, 938, 879, 851,
3
77
6
À1
1
798, 725 cm
J=6.5 Hz, CH ), 1.17±1.40 (40H, m, CH ), 1.61 (4H, m,
.
H NMR (CDCl ) d: 0.88 (6H, t,
3
1,3-Distearoyl-2-(4-iodobutanoyl)glycerol (5Cb). To a
stirred solution of sodium iodide (1.56 g, 10.5 mmol) in
anhydrous methyl ethyl ketone (11 mL) was added 4Cb
3
2
OCOCH CH ), 2.02 (8H, m, CH CHˆCHCH ), 2.12
2
2
2
2
(2H, m, COCH CH CH N), 2.33 (4H, t, J=7.5 Hz,
2 2 2
(0.83 g, 1.14 mmol). The stirring was continued for 48 h
at room temperature and the reaction mixture was con-
OCOCH ), 2.54 (2H, t, J=6.5 Hz, COCH CH CH N),
2 2 2 2
3.48 (9H, s, N(CH ) ), 3.75 (2H, m, COCH CH CH N),
3 3 2 2 2
centrated. The residue was dissolved in CHCl , washed
3
4.13, 4.40 (4H, ABX, J=5.5, 4.5 Hz, CH CHCH ), 5.16
2 2
with H O, 10% NaHSO , H O, 5% NaHCO , H O and
2
sat. NaCl. The organic layer was dried (MgSO ) and
4
(1H, m, CH CHCH ), 5.34 (4H, m, CH CHˆCHCH ).
2
3
2
3
2
2
2
2
1
3
C NMR (CDCl ) d: 173.40, 171.23, 130.01, 129.67,
3
concentrated. The residue was recrystallized from
hexane AcOEt (20:1) to a€ord colorless crystals 5Cb
70.39, 61.58, 53.78, 34.00, 31.88, 29.72, 29.67, 29.49,
29.29, 29.17, 29.09, 29.06, 27.19, 27.14, 24.82, 22.66, 18.31,
+
(
0.82 g, 88%).
14.11. MS (EI) m/z: 733 (M ÀCH I, 100), 265 (5.7), 142
3
(55.8). Anal. calcd for C H NO I: C, 63.06; H, 9.89; N,
1.60; I, 14.19. Found: C, 62.92; H, 9.60; N, 1.51; I, 14.14.
46 86 6
ꢀ
733, 1652, 1378, 1471, 1378, 1174, 1108, 1066 cm . H
Mp 61±62 C (hexane±AcOEt). IR ꢀ (KBr): 2914, 2849,
1
À1
1
NMR (CDCl ) d: 0.88 (6H, t, J=6.5 Hz, CH ), 1.18±
N-[4-[2-(1,3-Dioleoyloxy)propoxycarbonyl]butyl]-N,N,N-
trimethylammonium iodide (1Ac). Compound 1Ac was
prepared as described for 1Aa using 5Ac (0.27 g,
3
3
1
.36 (56H, m, CH ), 1.61 (4H, m, OCOCH CH ), 2.13
2 2 2
(
OCOCH ), 2.48 (2H, t, J=7.0 Hz), 3.24 (2H, t,
2H, m, COCH CH CH I), 2.32 (4H, t, J=7.5 Hz,
2 2 2
0.33 mmol). The residue was puri®ed by SiO column
2
2
J=7.0 Hz, COCH CH CH I), 4.13, 4.31 (4H, ABX,
2
chromatography (CHCl ±MeOH, 20:1) to a€ord a
3
2
2
J=6.0, 4.5 Hz, CH CHCH ), 5.27 (1H, m,
2
white wax 1Ac (0.217 g, 75%).
2
1
3
CH CHCH ). C NMR (CDCl ) d: 173.30, 171.43,
2
2
3
ꢀ
9
2
6.10, 69.38, 61.96, 34.68, 34.04, 31.93, 29.71, 29.49,
9.36, 29.27, 29.11, 28.29, 24.85, 22.70, 14.14, 5.03. MS
Mp 46.5±50 C. IR ꢀ (KBr): 2924, 2853, 1739, 1653,
À1
1
1457, 1418, 1373, 1173, 1096 cm . H NMR (CDCl ) d:
3
+
(EI) m/z: 820 (M , 2.9), 607 (100), 537 (33.1), 341
(17.4), 267 (30.0), 271 (16.2), 197 (37.4). Anal. calcd for
0.88 (6H, t, J=7.0 Hz, CH ), 1.18±1.40 (40H, m, CH ),
1.61 (4H, m, OCOCH CH ), 1.78 (2H, m,
2 2
3
2
C H O I: C, 62.91; H, 9.94; I, 15.46. Found: C, 63.05;
4
COCH CH (CH ) N), 1.90 (2H, m, CO(CH ) CH -
2 2 2 2 2 2 2
3
81
6
H, 9.71; I, 15.13.
CH N), 2.00, 2.33 (4H each, m, CH CHˆCHCH ), 2.33
2
2
2
(
4H, t, J=7.5 Hz, OCOCH ), 2.45 (2H, t, J=6.0 Hz,
2
N-[2-(1,3-Dioleoyloxy)propoxycarbonylmethyl]-N,N,N-
trimethylammonium chloride (1Aa). Excess amount of
anhydrous trimethylamine was added to a solution of
OCOCH CH ), 3.46 (9H, s, N(CH ) ), 3.71 (2H, m,
2 2 3 3
CO(CH ) CH CH N), 4.11, 4.41 (4H, ABX, J=5.5,
2
2
2
2
4.5 Hz, CH CHCH ), 5.17 (1H, m, CH CHCH ), 5.34
2 2 2 2
ꢀ
13
4
Aa (1.40 g, 2.01 mmol) in dry Et O (4 mL) at 0 C. The
2
(4H, m, CH CHˆCHCH ). C NMR (CDCl ) d:
2
2
3
reaction mixture was stirred for 12 h at the same tem-
perature and concentrated. The residue was puri®ed by
SiO column chromatography (hexane) to give a white
173.39, 171.88, 130.01, 129.65, 69.72, 66.51, 61.64,
53.80, 34.04, 32.80, 31.86, 29.72, 29.67, 29.49, 29.29,
29.15, 29.09, 29.06, 27.19, 27.14, 24.85, 22.64, 22.18,
2
+
wax 1Aa (1.01 g, 66%).
20.95, 14.09. MS (EI) m/z: 747 (M ÀCH I, 100), 265
3
(5.1), 142 (53.8), 128 (64.3). Anal. calcd for
C H NO I 1/2H O: C, 62.78; H, 9.89; N, 1.56; I,
14.11. Found: C, 62.82; H, 9.63; N, 1.59; I, 13.89.
ꢀ
ꢁ
Mp 35±39 C. IR ꢀ (KBr): 2924, 2853, 2062, 1746, 1634,
4
7
88
6
2
À1
1
1
464, 1399, 1255, 1196, 1091, 1008 cm
.
H NMR
(
(
(
CDCl ) d: 0.88 (6H, t, J=6.5 Hz, CH , 1.00±1.46
3
3)
40H, m, CH ), 1.58 (4H, m, OCOCH CH ), 2.00, 2.02
4H each, m, CH CHˆCHCH ), 2.35 (4H, t, J=7.5 Hz,
N - [2 - (1,3 - Dielaidoyloxy)propoxycarbonylmethyl] -
N,N,N-trimethylammonium chloride (1Ba). Compound
1Ba was prepared as described for 1Aa using 4Ba
2
2
2
2
2
OCOCH ), 3.67 (9H, s, N(CH ) ), 4.11, 4.48 (4H, ABX,
2
3 3
J=5.5, 4.5 Hz, CH CHCH ), 4.92 (2H, s, COCH N),
2
(0.60 g, 0.86 mmol). The residue was washed with Et O
2
2
2
5
.22 (1H, m, CH CHCH ), 5.34 (4H, m,
2
and dried under reduced pressure to a€ord a white
powder 1Ba (0.57 g, 88%).
2
1
3
CH CHˆCHCH ).
C NMR (CDCl ) d: 173.51,
2
2
3
1
3
2
8
64.33, 130.01, 129.67, 72.30, 63.09, 61.21, 54.34, 33.95,
1.88, 29.74, 29.69, 29.60, 29.49, 29.29, 29.17, 29.09, 27.21,
ꢀ
Mp 71±75 C. IR ꢀ (KBr): 2917, 2851, 1742, 1629, 1470,
+
À1
1
7.15, 24.78, 22.66, 14.09. MS (EI) m/z: 705(M ÀCH Cl,
1277, 1163, 1026 cm . H NMR (CDCl ) d: 0.87 (6H, t,
3
3
1.6), 602 (10.0), 265 (26.6), 55 (100). Anal. calcd for
.
J=6.5, CH ), 1.15±1.40 (40H, m, CH ), 1.60 (4H, m,
OCOCH CH ), 1.94, 1.96 (4H each, m, CH CHˆCH-
3
2
C H NO Cl 2H O: C, 66.68; H, 10.43; N, 1.77; Cl, 4.47.
2
44
82
6
2
2
2
Found: C, 66.96; H, 10.72; N, 1.99; Cl, 4.27.
CH ), 2.34 (4H, t, J=7.5 Hz, OCOCH ), 3.66 (9H, s,
2 2
N(CH ) ), 4.10, 4.47 (4H, ABX, J=5.5, 4.0 Hz,
3
3
N - [3 - [2 - (1,3 - Dioleoyloxy)propoxycarbonyl]propyl] -
N,N,N-trimethylammonium iodide (1Ab). Compound
CH CHCH ), 4.89 (2H, s, COCH N), 5.21 (1H, m,
2 2 2
1
3
CH CHCH ), 5.37 (4H, m, CH CHˆCHCH ).
C
2
2
2
2

S. Obika et al. / Bioorg. Med. Chem. 9 (2001) 245±254
253
NMR (CDCl ) d: 173.46, 164.31, 130.44, 130.10, 72.20,
(6H, t, J=5.5 Hz, CH , 1.16±1.38 (56H, m, CH ), 1.61
3) 2
(4H, m, OCOCH CH ), 2.12 (2H, m, COCH2-
2 2
CH CH N), 2.33(4H, t, J=7.5 Hz, OCOCH ), 2.54
2 2 2
(2H, t, J=6.5 Hz, COCH CH CH N), 3.47 (9H, s,
2 2 2
3
6
2
3.06, 61.19, 54.23, 33.93, 32.56, 31.88, 29.65, 29.45,
9.31, 29.24, 29.15, 29.09, 28.93, 24.78, 22.64, 14.07. MS
+
(
(
EI) m/z: 706 (M ÀCH Cl, 6.9), 602 (1.1), 265 (1.9), 58
3
ꢁ
100). Anal. calcd for C H NO Cl 1/2H O: C, 69.03;
4
N(CH ) ), 3.73 (2H, m, COCH CH CH N), 4.12, 4.41
3 3 2 2 2
4
82
6
2
H, 10.93; N, 1.83; Cl, 4.63. Found: C, 68.78; H, 10.63;
N, 2.02; Cl, 4.59.
(4H, ABX, J=5.5, 4.0 Hz, CH CHCH ), 5.16 (1H, m,
2 2
1
3
CH CHCH ). C NMR (CDCl ) d: 173.40, 171.19,
2
2
3
96.07, 70.33, 65.54, 61.57, 53.77, 34.00, 31.88, 29.67,
29.45, 29.33, 29.24, 29.08, 24.84, 22.64, 18.30, 14.09. MS
N - [3 - [2 - (1,3 - Dielaidoyloxy)propoxycarbonyl]propyl] -
N,N,N-trimethylammonium iodide (1Bb). Compound
+
(EI) m/z: 737 (M ÀCH I, 4.4), 341 (1.4), 267 (2.2), 142
3
1Bb was prepared as described for 1Aa using 5Bb
0.79 g, 0.97 mmol). The residue was washed with Et O
(23.1), 114 (14.9), 58 (100). Anal. calcd for C H NO I:
4
6
90
6
(
and dried under reduced pressure to a€ord a white
C, 62.78; H, 10.30; N, 1.59; I, 14.41. Found: C, 62.49;
H, 10.03; N, 1.63; I, 14.48.
2
powder 1Bb (0.67 g, 78%).
Biological assay
ꢀ
363, 1252, 1176, 1116, 1065, 1022 cm
Mp 74±77 C. IR ꢀ (KBr): 2920, 2850, 1754, 1465, 1416,
À1
1
1
.
H NMR
Prepartion of cationic liposomes and transfection
experiments were carried out according to the procedure
previously reported.
(
(
(
CDCl ) d: 0.88 (6H, t, J=6.5 Hz, CH ), 1.19±1.50
3
3
1
2
40H, m, CH ), 1.61 (4H, m, OCOCH CH ), 1.95, 1.97
4H each, m, CH CHˆCHCH ), 2.14 (2H, m,
2
2
2
2
2
COCH CH CH N), 2.33 (4H, t, J=7.5 Hz, OCOCH ),
2
2
2
2
2
N(CH ) ), 3.76 (2H, m, COCH CH CH N), 4.13, 4.39
.54 (2H, t, J=7.0 Hz, COCH CH CH N), 3.49 (9H, s,
Acknowledgement
2
2
2
3
3
2
2
2
(
4H, ABX, J=5.0, 4.5 Hz, CH CHCH ), 5.17 (1H, m,
2
Part of this work was supported by a Grant-in-Aid for
Scienti®c Research (B), No. 11470469, from Japan
Society for the Promotion of Science.
2
1
3
CH CHCH ), 5.38 (4H, m, CH CHˆCHCH ).
C
2
2
2
2
NMR (CDCl ) d: 173.30, 171.12, 130.39, 130.06, 70.23,
3
6
2
2
2
5.52, 61.55, 53.75, 33.96, 32.51, 32.47, 31.81, 29.71,
9.56, 29.40, 29.38, 29.22, 29.09, 29.04, 28.99, 28.90,
+
4.76, 22.57, 18.30, 14.02. MS (EI) m/z: 733 (M ÀCH I,
3
1.6), 339 (1.1), 265 (1.0), 142 (75.9), 58 (100). Anal.
calcd for C H NO I: C, 63.06; H, 9.89; N, 1.60; I,
4
References and Notes
6
86
6
1
4.49. Found: C, 62.77; H, 9.65; N, 1.63; I, 14.52.
1. Kasid, A.; Morecki, S.; Aebersold, P.; Cornetta, K.; Culver,
K.; Freeman, S.; Director, E.; Lotze, M. T.; Blaese, R. M.;
N - [2 - (1,3 - Distearoyloxy)propoxycarbonylmethyl] -
N,N,N-trimethylammonium chloride (1Ca). Compound
Anderson, W. F.; Rosenberg, S. A. Proc. Natl. Acad. Sci. USA
990, 87, 473; Anderson, W. F. Science 1992, 256, 808; Mulli-
1
1
Ca was prepared as described for 1Aa using 4Ca
0.92 g, 1.31 mmol). The residue was washed with Et O
gan, R. C. Science 1993, 260, 926; Dunbar C. E. Annu. Rev.
Med. 1996, 47, 11.
(
and dried under reduced pressure to a€ord a white
2
2
1
1
. Vile, R. G.; Tuszynski, A.; Castleden, S. Mol. Biotechnol.
996, 5, 139; Moolten, F. L.; Cupples, L. A. Hum. Gene Ther.
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powder 1Ca (0.94 g, 94%).
ꢀ
471, 1398, 1255, 1236, 1196, 1182 cm
Mp 99.5±101.5 C. IR ꢀ (KBr): 2915, 2848, 1740, 1647,
Hum. Gene Ther. 1991, 2, 5.
3. Lee, R. J.; Huang, L. Crit. Rev. Ther. Drug Carrier Syst.
1997, 14, 173.
4. Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan,
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À1
1
1
.
H NMR
(
(
CDCl ) d: 0.88 (6H, t, J=6.0 Hz, CH ), 1.15±1.36
3
3
56H, m, CH ), 1.60 (4H, m, OCOCH CH ), 2.35 (4H,
2
2
2
t, J=8.0 Hz, OCOCH ), 3.63 (9H, s, N(CH ) ), 4.07,
2
3 3
4
.55 (4H, ABX, J=7.0, 5.0 Hz, CH CHCH ), 4.92 (2H,
2 2
1
3
s, CH ), 5.20 (1H, m). C NMR (CDCl ) d: 173.49,
2
3
5. For reviews: Miller, A. D. Angew. Chem. Int. Ed. 1998, 37,
1768; Bennett, M. J.; Aberle, A. M.; Balasubramaniam, R. P.;
1
2
64.33, 72.26, 63.07, 61.21, 54.29, 33.96, 31.90, 29.67,
9.47, 29.33, 29.26, 29.11, 24.80, 22.66, 14.09. MS (EI)
Malone, J. G.; Nantz, M. H.; Malone, R. W. J. Liposome Res.
1996, 6, 545; Felgner, P. L. J. Liposome Res. 1993, 3, 3; Brig-
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Tsai, Y. J.; Border, R.; Ramsey, P.; Martin, M.; Felgner, P. L.
J. Biol. Chem. 1994, 269, 2550.
+
m/z: 709 (M ÀCH Cl, 6.9), 605 (5.4), 341 (7.2), 267
3
.
20.1), 85 (100). Anal. calcd for C H NO Cl 1/2 H O:
44 86 6 2
(
C, 68.67; H, 11.39; N, 1.82; Cl, 4.61. Found: C, 68.72;
H, 11.31; N, 1.92; Cl, 5.04.
7
1
8
1
9
. Leventis, R.; Silvius, J. R. Biochim. Biophys. Acta 1990,
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. Gao, X.; Huang, L. Biochem. Biophys. Res. Commun. 1991,
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N - [3 - [2 - (1,3 - Distearoyloxy)propoxycarbonyl]propyl] -
N,N,N-trimethylammonium iodide (1Cb). Compound
1
Cb was prepared as described for 1Aa using 5Cb
0.82 g, 1.00 mmol). The residue was washed with Et O
(
and dried under reduced pressure to a€ord a white
2
Proc. Natl. Acad. Sci. USA 1989, 86, 6982.
0. Demeneix, B. A.; Abdel-Taweb, H.; Benoist, C.; Seugnet,
I.; Behr, J.-P. BioTechniques 1994, 16, 496.
powder 1Cb (0.50 g, 57%).
1
ꢀ
Mp 100±102 C. IR ꢀ (KBr): 2915, 2849, 1733, 1471,
11. Hawley-Nelson, P.; Ciccarone, V.; Gebeyehu, G.; Iessee,
J.; Felgner, P. L. Focus 1993, 1, 573.
À1
1
1
417, 1254, 1175, 1115 cm . H NMR (CDCl ) d: 0.88
3

254
S. Obika et al. / Bioorg. Med. Chem. 9 (2001) 245±254
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1
1
817.
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1
1
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1
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1