Synthesis of cationic cardiolipin analogues

Article abstract of DOI:10.1016/j.bioorg.2005.06.001

An approach was developed to synthesize a new class of cationic cardiolipin analogues containing two quaternary ammonium groups with tetra alkyl groups retaining "glycerol" moiety, the central core of the molecule. Cationic cardiolipin analogues were modified via introduction of either two or four oxyethylene groups to enhance the solubility in polar solvents. These newly synthesized cationic cardiolipin analogues can be applied to a broad range of drug delivery systems such as transfection reagents.

Full text of DOI:10.1016/j.bioorg.2005.06.001

Bioorganic Chemistry 33 (2005) 345–362
www.elsevier.com/locate/bioorg
Synthesis of cationic cardiolipin analogues
Krishnudu Kasireddy, Shoukath M. Ali, Moghis U. Ahmad,
Sreeti Choudhury, Pei-Yu Chien, Saifuddin Sheikh,
Imran Ahmad ¤
Research and Development Facility, NeoPharm, Inc., 1850 Lakeside Drive, Waukegan, IL 60085, USA
Received 1 March 2005
Available online 12 September 2005
Abstract
An approach was developed to synthesize a new class of cationic cardiolipin analogues con-
taining two quaternary ammonium groups with tetra alkyl groups retaining “glycerol” moiety,
the central core of the molecule. Cationic cardiolipin analogues were modiWed via introduction
of either two or four oxyethylene groups to enhance the solubility in polar solvents. These
newly synthesized cationic cardiolipin analogues can be applied to a broad range of drug deliv-
ery systems such as transfection reagents.
2005 Elsevier Inc. All rights reserved.
Keywords: Cationic lipids; Cardiolipin; Transfection; Gene delivery; Lipid-based formulations
1. Introduction
In recent years, a signiWcant research eVort has been devoted in developing new
cationic lipids to assist delivery of DNA [1], mRNA [2], siRNA [3], antisense oligonu-
cleotide [4], proteins [5], and antiviral [6,7] into human cells. Cationic liposomes are
recognized as an important carrier to deliver anionic species, such as genes or other
*
Corresponding author. Fax: +1 847 887 9281.
E-mail address: Imran@neophrm.com (I. Ahmad).
0045-2068/$ - see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2005.06.001

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K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
nucleic acids, to cells. Cationic liposomes are thought to interact electrostatically
with negatively charged nucleic acid sequences to form complexes that facilitate pen-
etration of these agents into cells. Thus, cationic lipids could play a role in delivering
anionic agents into target cells and tissues in the treatment of disease. Consequently,
a need has arisen for the development of a new class of cationic lipids that are less
toxic and synthetic methods that are simple and scalable. Since the Wrst description
by Felgner et al. [8] and their discovery of a potential cationic lipid such as N-[1-(2,3-
dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) for transfec-
tion, an increasing number of new cationic lipids of diVerent structures have been
synthesized [9] and their respective transfection activities in wide variety of cell types
have been reported.
Cationic lipids are composed of three parts (Fig. 1), a cationic head group, a lipo-
philic tail group, and a linker that tethers the hydrophilic head group and hydropho-
bic tail group. Cationic head groups include quaternary ammonium salt lipids,
lipoamines (primary, secondary, and tertiary amine lipids), and combinations of both
lipoamines and quaternary amines. Tail groups usually consist of saturated or unsat-
urated alkyl chains (12–18 carbons in length) or cholesteryl groups.
Commercially available transfection reagents include, DOTMA [8], 1,2-
dioleyloxy-3-(trimethylammonio)propane chloride (DOTAP) [10], N,N-dimethyl-N-
[2-(sperminecarboxamido)ethyl]-2,3-bis(dioleyloxy)-1-propanaminium pentahydro-
chloride (DOSPA) [11], 1,3-dioleoyloxy-2-(6-carboxyspermyl)propyl amide (DOSPER)
[12], dimethyldioctadecylammonium bromide (DDAB) [13], N,N-dioleyl-N,N-dime-
thylammonium chloride (DODAC) (Fig. 2), and cationic cardiolipin analogue 3 [14]
(PCL-2) (Fig. 4). These cationic lipids, with combination of helper lipid like 1,2-diol-
eoyl phosphatidylethanolamine (DOPE) or 1,2-dioleoyl-sn-glycero-3-phosphocho-
line (DOPC), showed transfection activity.
The structure of cationic lipids is an important determinant for their transfection
activity. By comparing the diVerent structures and their transfection activities in the
same family or diVerent families of lipids, some common conclusions can be made.
The hydrocarbon chain length has eVects on transfection activity in cell culture. The
transfection activity of cationic lipids of varying chain length is usually in the order
C14:0 (myristyl)> C16:0 (palmityl) > C18:0 (stearyl) [15]. It is possible that shorter
hydrocarbon chains decrease the rigidity of bilayer and favor a higher intermem-
brane transfer rate and lipid mixing, resulting in potential disruption of the endo-
some and consequent DNA escape from endosomal degradation [16]. Structural
variations at the linker region such as length, the speciWc type of chemical bonds, and
the relative position of the hydrocarbon chains can aVect the transfection eYciency,
Spacer
Hydrocarbon chains
Head
Fig. 1. Basic structure of cationic lipids.

K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
347
O
O
+
+
N
N
Br
Cl
DOTMA
DDAB
O
O
Cl
N
O
O
+
N
Cl
DODAC
DOTAP
O
O
O
Cl
N
H N
+
2
N
H
N
H
N
H
O
O
H N
2
DOSPA
O
O
O
H N
2
N
H
N
H
-
4 CH COO
N
H
3
DOSPER
H N
2
Cl
N
Cl
N
TODMAC6
Fig. 2. Chemical structures of cationic lipids.
biodegradability, and stability of the cationic lipids. Cationic lipid such as DOTMA,
which contains an ether linkage, was shown to have much greater in vivo transfection
eYciency [17] than the corresponding ester analogue DOTAP. Notwithstanding the
lower transfection eYciency of DOTAP, it was used for in vivo gene delivery on
human subjects due to its low cytotoxicity while DOTMA is used only for in vitro
studies. The quaternary ammonium lipids (DOTMA or DOTAP) of this family were
modiWed by replacing the methyl group with the alkylene alcohol or alkylene tail
group with alkyl group to give 1,2-dioleyloxypropyl-3-dimethylhydroxyethyl ammo-
nium [18] (DORIE) or 1,2-dimyristyloxy propyl-3-dimethylhydroxyethyl ammonium
(DMRIE), respectively. The hydroxyl group in DORIE or DMRIE may increase the
interaction of DNA with lipids or improve the interaction of the DNA/liposome
complexes with cellular membranes, leading to greater in vitro and in vivo activity
than DOTMA or DOTAP. N,N,NЈ,NЈ-Tetraoleyl-N,N-dimethyl-1,6-hexanediammo-
nium chloride [19] (TODMAC6, divalent and tetra alkyl) was shown to possess bet-
ter transfection properties compared to DODAC (monovalent and dialkyl).
Despite numerous investigations of cationic lipids in gene therapy, most of the
known cationic lipids possess certain toxicities. Recognizing the need for development
of new cationic lipids to improve gene therapy and drug delivery, we undertook a pro-
gram to design and synthesize a new class of dimeric cationic lipids (Fig. 3). It has been
suggested that the ability of cationic lipids to facilitate intracellular delivery is related to
their ability to induce non-bilayer hexagonal HII phase in combination with anionic lip-
ids at endosomal stage, and that the most potent cationic lipids are those that are the
most eVective inducers of HII phase organization [20]. It is known that the ability of lip-
ids to adopt the HII phase is related to their dynamic molecular shapes. In general, a

348
K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
C
C
H
O
O
OC
OC
H
14 29
14 29
H
H
14 29
14 29
+
+
N
N
O
O
O
O
-
-
p
Br
Br
r
q
r
p
q
OH
p = 0 q = 1
r = 0
2
p = 0 q = 1 r = 1
p = 1 q = 1 r = 0
p = 1 q = 1 r =1
3
4
5
Fig. 3. General structure of cationic cardiolipin analogue.
O
O
O
O
O
O
O
O
C
C
H
C
C
H
13 27
13 27
H
O
P
OH
1
O
P
H
13 27
13 27
O
O
+
O
O
+
-
-
O X
O X
Cardiolipin
OC
OC
H
C
H O
14 29
14 29
C
H
O
OC
H
14 29
14 29
H
14 29
C
14 29
H
O
OH
OC
14 29
H
C
H O
14 29
OH
+
+
+
+
O
O
N
N
N
N
-
-
Br
Br
-
-
Br
Br
3 (PCL-2)
2 (PCL-1)
C
H O
OC
OC
H
14 29
C
H
O
O
OC
H
14 29
14 29
14 29
C
14 29
H O
H
14 29
OH
OC
14 29
H
C
H
+
N
Br
+
14 29
+
+
N
N
N
O
O
O
O
-
-
O
-
O
-
Br
Br
Br
OH
4 (PCL3)
5 (PCL-4)
Fig. 4. Chemical structures of cardiolipin and cationic cardiolipin analogues.
lipid with a small cross-sectional area in the headgroup region and a larger acyl chain
cross-sectional area exhibits a “cone” shape compatible with HII phase organization
and are known to enhance the transfection eYciency [21]. One method to increase the
“cone” shape character is to create a dimer of cationic lipids joined together in the
headgroup region by a spacer with reasonable length. Recently, Cullis [19] utilized this
strategy to synthesize a novel dimer cationic lipid and achieved improved transfection
activity as compared with monomer lipid. In the present study, we adopted similar
strategy to design novel cationic cardiolipin (dimeric cationic lipid) analogues. We
developed synthetic methods for cationic cardiolipin (dimeric cationic lipid) analogues.
Cardiolipin 1 (glycerol-bridged dimeric phosphatidic acid) [22] constitutes a class of
complex phospholipids that occur mainly in the heart and skeletal muscles, showing
high metabolic activity. Cardiolipin has two negatively charged phosphate groups,
which were replaced with quaternary ammonium groups to provide cationic cardiolipin
analogue 2 [14] (Fig. 4). Cationic cardiolipin analogue 2 was further modiWed with the

K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
349
addition of oxyethylene group on both sides of the central glycerol moiety to obtain
cationic cardiolipin analogue 3 [14]. Exchanging the position of oxa group and the qua-
ternary ammonium group will provide cationic cardiolipin analogue 4. The cationic
cardiolipin analogue 3 was modiWed with the addition of oxyethylene group on both
sides of the quaternary ammonium group to yield spacer cationic cardiolipin analogue
5 (Fig. 4). The cationic cardiolipin analogues were designed accommodating some key
features from known cationic lipids such as the presence of two positive charges and
four saturated hydrocarbon chains with ether linkages (C14:0, myristyl). The central
glycerol units in all these cationic lipids were retained to mimic the naturally occurring
cardiolipin. We anticipated that these chemical modiWcations would be helpful for an
eYcient transfection.
2. Materials and methods
2.1. General
Melting points were determined at atmospheric pressure and uncorrected. 1H NMR
spectra were recorded on Varian Inova NMR spectrometer at 300 and 500MHz. 13C
NMR spectra were recorded on Varian Inova (500MHz) NMR spectrometer at
1
125MHz. H chemical shifts are reported in ppm from internal tetramethylsilane. 13C
chemical shifts are reported in ppm relative to CDCl3 (77.0ppm). Mass spectral analyses
[electron spray ionization (ESI)] were carried out on Triple Quadruple LC/MS/MS mass
spectrometer API 4000 (Applied Biosystems, Foster City, CA). Infrared (IR) spectra
were recorded on a Nicolet Nexus 470 FT-IR. Samples were prepared by ATR method.
Thin-layer chromatography (TLC) was carried out on Merck silica gel 60 F254 plates
(250ꢀm) and developed with the appropriate solvents. The TLC spots were visualized
either by UV light or by heating plates sprayed with a solution of phosphomolybdic acid
(5% ethanolic solution). Flash column chromatography was carried out on silica gel
(230–400 mesh). All chemicals and anhydrous solvents were purchased from Aldrich
Chemical (Milwaukee, WI). All of the extracts were dried over anhydrous Na2SO4.
2.2. Synthesis of bromo intermediates
2.2.1. 1,3-Dibromo-2-O-benzyl glycerol (7)
To a solution of 2-O-benzyl glycerol (6) (20g, 109 mmol) in anhydrous dichloro-
methane (275mL) under argon atmosphere at 0°C was added triphenylphosphine
(64.4 g, 230mmol) followed by carbon tetrabromide (76.3 g, 230 mmol). The reaction
mixture was stirred at 0°C for 2 h. The reaction mixture was diluted with water
(300 mL) and the organic layer was separated, dried over sodium sulfate. The organic
layer was concentrated under reduced pressure and crude product was puriWed by
column chromatography over a silica gel (230–400 mesh) with 1–5% ethyl acetate in
hexane to obtain 1,3-dibromo-2-O-benzyl glycerol (7) (26 g, 77%) as colorless oil.
TLC (SiO2) hexane/ethyl acetate (9:1) Rf »0.80. 1H NMR (CDCl3, 500 MHz): ꢀ 3.52–
3.62 (m, 4H), 3.78–3.82 (m, 1H), 4.65 (s, 2H), 7.30–7.38 (m, 5H).

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K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
2.2.2. 1,3-Dibromopropane-2-ol (8)
1,3-Dibromo-2-O-benzyl glycerol (7) (23g, 74.6 mmol) was dissolved in ethanol
(100mL) and hydrogenated with 10% palladium on carbon (0.5g) for 4h at 50 psi.
After Wltration of the catalyst, the solution was evaporated under reduced pressure.
The crude material was subjected to silica-gel column chromatography (230–400
mesh) and eluted with 1–20% ethyl acetate in hexane to obtain 1,3-dibromopropane-2-
1
ol (8) (15g, 93%) as colorless oil. TLC (SiO2) hexane/ethyl acetate (3.5:1) Rf »0.5. H
NMR (CDCl3, 500MHz): ꢀ 2.66 (br s, 1H, OH), 3.58–3.59 (m, 4H), 4.01–4.03 (m, 1H).
2.2.3. 1,3-Bis[(2-ethoxy tetrahydro-2H-pyran)]-2-O-benzyl glycerol (9)
To a stirred suspension of sodium hydride (59.3g, 1.48mol, 60% in oil) in anhy-
drous dimethylformamide (300mL) under argon atmosphere at 0 °C, a solution of 2-
O-benzyl glycerol (6) (90 g, 0.49mol) in dimethylformamide (700 mL) was added over
a period of 2 h maintaining the temperature below 15°C. After stirring at room tem-
perature for 2 h, 2-(2-bromoethoxy)tetrahydro-2H-pyran (310 g, 1.48 mol) was added
at 0°C over a period of 3h maintaining the temperature below 10°C. The reaction
mixture was stirred at room temperature for 12 h, then cooled to 0°C, and ice water
was added slowly to quench the excess sodium hydride. The reaction mixture was
concentrated under reduced pressure to remove maximum DMF and the crude solu-
tion was diluted with water (1 L) and extracted with ethyl acetate (2£ 500 mL). The
organic layer was washed with aqueous saturated sodium chloride (500mL) and
dried over sodium sulfate. The solvent was concentrated under reduced pressure. The
crude product was puriWed by column chromatography over a silica gel (230–400
mesh) with 10–30% ethyl acetate in hexane to obtain 1,3-bis-[(2-ethoxy tetrahydro-
2H-pyran)]-2-O-benzyl glycerol (9) (154 g, 71%) as colorless oil. TLC (SiO2) hexane/
ethyl acetate (3:2) Rf » 0.40. 1H NMR (CDCl3, 300MHz): ꢀ 1.41–1.82 (m, 12H), 3.41–
3.98 (m, 17H), 4.61 (br s, 2H), 4.78 (s, 2H, OCH2Ph), 7.24–7.45 (m, 5H, Ph-H).
2.2.4. 3,7-Dioxa-5-O-benzyl-1,9-nonanediol (10)
To a solution of 1,3-bis[(2-ethoxy tetrahydro-2H-pyran)]-2-O-benzyl glycerol (9)
(50g, 0.11 mol) in methanol (500 mL) was added 1 M HCl in ether (5mL) and stirred
at room temperature for 2h. The reaction mixture was neutralized with solid sodium
bicarbonate until the solution became neutral. The reaction mixture was Wltered and
concentrated under reduced pressure. The crude product was dissolved in ethyl ace-
tate (1 L), washed with water (100mL), and dried over sodium sulfate. The organic
layer was concentrated under reduced pressure and the crude product was puriWed by
column chromatography over a silica gel (70–230 mesh) eluting with ethyl acetate,
followed by 5% methanol in ethyl acetate to obtain 3,7-dioxa-5-O-benzyl-1,9-non-
anediol (10) (27g, 88%) as colorless oil. TLC (SiO2) ethyl acetate Rf » 0.10. 1H NMR
(CDCl3, 300 MHz): ꢀ 2.58 (br s, 2H, OH), 3.50–3.81 (m, 13H), 4.68 (s, 2H, OCH2Ph),
7.21–7.42 (m, 5H, Ph-H).
2.2.5. 1,9-Dibromo-3,7-dioxa-5-O-benzylnonane (11)
To a solution of 3,7-dioxa-5-O-benzyl-1,9-nonanediol (10) (27 g, 0.1 mol) in anhy-
drous dichloromethane (400mL) under argon atmosphere at 0 °C, was added triphe-

K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
351
nylphosphine (65.5g, 0.25mol) followed by carbon tetrabromide (79.4 g, 0.24 mol).
The reaction mixture was stirred at 0 °C for 2h. The reaction mixture was diluted
with water (300mL) and the organic layer was separated, and dried over sodium sul-
fate. The organic layer was concentrated under reduced pressure and crude product
was puriWed by column chromatography over a silica gel (70–230 mesh) with 20%
ethyl acetate in hexane to obtain 1,9-dibromo-3,7-dioxa-5-O-benzylnonane (11) (36 g,
1
91%) as colorless oil. TLC (SiO2) hexane/ethyl acetate (3:2) Rf »0.60. H NMR
(CDCl3, 500MHz): ꢀ 3.45 (t, J D 6.5Hz, 4H, CH2Br), 3.60–3.67 (m, 4H, OCH2), 3.72–
3.82 (m, 5H), 4.70 (s, 2H, OCH2Ph), 7.28–7.38 (m, 5H, Ph-H).
2.2.6. 1,3-bis-(2-Bromoethoxy)propane-2-ol (12)
1,9-Dibromo-3,7-dioxa-5-O-benzylnonane (11) (36 g, 90.90 mmol) was dissolved in
ethanol (110 mL) and hydrogenated with 10% palladium on carbon (3.6g) for 2 h at
50psi. After Wltration of the catalyst, the solution was evaporated under reduced
pressure. The crude material was subjected to silica-gel column chromatography (70–
230 mesh) and eluted with 60% ethyl acetate in hexane to obtain 1,3-bis-(2-bromo-
ethoxy)propane-2-ol (12) (26 g, 94%) as colorless oil. TLC (SiO2) hexane/ethyl acetate
1
(1:1) Rf » 0.30. H NMR (CDCl3, 500 MHz): ꢀ 2.08 (s, 1H, OH), 3.45 (t, J D6.0Hz,
4H, CH2Br), 3.54–3.64 (m, 4H, OCH2), 3.63–3.85 (m, 4H, OCH2), 3.96–4.02 (m, 1H).
2.3. Synthesis of tertiary amine intermediates
2.3.1. (S)-1,2-bis-Tetradecyloxy-3-O-benzylpropane (14)
To a stirred suspension of sodium hydride (54.5 g, 1.36mol, 60% in oil) in anhy-
drous dimethylformamide (220 mL) under argon atmosphere at 0°C, a solution of
(S)-1-O-benzyl glycerol (13) (62 g, 0.34 mol) in dimethylformamide (400 mL) was
added over a period of 1 h maintaining the internal temperature below 20 °C. After
stirring at room temperature for 2 h, tetradecyl bromide (377.4 g, 1.36 mol) was added
at 0°C over a period of 2 h. After complete addition, the reaction mixture was stirred
for 2h at room temperature and the temperature was gradually increased to 70 °C,
and then stirred for 5h. The reaction mixture was cooled to 0 °C and quenched with
few drops of cold water. The mixture was diluted with saturated ammonium chloride
(500 mL). The aqueous layer was extracted with ethyl acetate (1L) and washed with
water (3£ 1 L), and dried over sodium sulfate. The organic layer was concentrated
under reduced pressure and the crude product was puriWed by column chromatogra-
phy over a silica gel (70–230 mesh) eluting with 2–10% ethyl acetate in hexane to
obtain (S)-1,2-bis-tetradecyloxy-3-O-benzylpropane (14) (146 g, 75%) as colorless oil.
TLC (SiO2) hexane/ethyl acetate (1:9) Rf »0.53. 1H NMR (CDCl3, 300 MHz): ꢀ 0.88
(t, J D 6.7Hz, 6H), 1.25 (br s, 44H), 1.49–1.58 (m, 4H), 3.41 (t, J D 6.5Hz, 2H), 3.42–
3.61 (m, 7H), 4.54 (m, 2H, OCH2Ph), 7.23–7.32 (m, 5H, Ph-H).
2.3.2. (R)-1,2-bis-Tetradecyloxypropan-3-ol (15)
A solution of (S)-1,2-bis-tetradecyloxy-3-O-benzylpropane (14) (70g, 0.12mmol)
was dissolved in ethyl acetate (280mL) and hydrogenated with 10% palladium on
carbon (3g) for 12 h at 50 psi. After Wltration of the catalyst, the solution was

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K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
evaporated under reduced pressure. The residue was dissolved in hot ethanol
(500mL) and kept at ¡20 °C overnight. The precipitated solid was Wltered and dried
under vacuum to obtain (R)-1,2-bis-tetradecyloxypropan-3-ol (15) (54g, 92%) as a
white solid. Mp 44–45°C. TLC (SiO2) hexane/ethyl acetate (1:9) Rf »0.17. 1H NMR
(CDCl3, 300 MHz): ꢀ 0.88 (t, J D6.7 Hz, 6H), 1.25 (br s, 44H), 1.59–1.57 (m, 4H), 2.2
(t, J D 5.7 Hz, 1H, OH), 3.37–3.73 (m, 9H).
2.3.3. (S)-1,2-bis-Tetradecyloxy-3-bromopropane (16)
To a solution of (R)-1,2-bis-tetradecyloxypropan-3-ol (15) (52g, 0.1 mol) in anhy-
drous dichloromethane (280mL) under argon atmosphere at 0°C triphenylphos-
phine (35.1g, 0.13 mol) was added. A solution of carbon tetrabromide (46.2 g,
0.13mol) in dichloromethane (240 mL) was added to the reaction mixture dropwise
in a period of 1 h and further stirred at 0°C for 3 h. The reaction mixture was diluted
with water (500mL) and the organic layer was separated, and dried over sodium
sulfate. The organic layer was concentrated under reduced pressure and the crude
product was puriWed by column chromatography over a silica gel (230–400 mesh)
with 1–5% ethyl acetate in hexane to obtain (S)-1,2-bis-tetradecyloxy-3-bromopro-
pane (16) (53 g, 90%) as colorless oil. TLC (SiO2) hexane/ethyl acetate (1:9) Rf » 0.72.
1H NMR (CDCl3, 300MHz): ꢀ 0.88 (t, J D 6.7Hz, 6H), 1.25 (br s, 44H), 1.51–1.61 (m,
4H), 3.39–3.61 (m, 9H).
2.3.4. (R)-1,2-bis-Tetradecyloxy-3-dimethylamino propane (17)
(S)-1,2-Bis-tetradecyloxy-3-bromopropane (16) (50g, 0.09mol) was dissolved in a
2 M methanolic solution of dimethylamine (400mL) in a screw-top pressure bottle.
The pressure bottle was sealed and heated in an oil bath at 88–90 °C for 60 h while
stirring. The pressure bottle was cooled to room temperature before it was opened.
The solution was concentrated under reduced pressure. The crude residue was dis-
solved in ethyl acetate (500mL) and washed with water (500mL). The organic layer
was concentrated under reduced pressure and puriWed by column chromatography
over a silica gel (230–400 mesh) with 5–20% ethyl acetate in hexane as eluent to
obtain (R)-1,2-bis-tetradecyloxy-3-dimethylamino propane (17) (41 g, 88%) as light
1
colored oil. TLC (SiO2) methanol/chloroform (1:9) Rf » 0.51. H NMR (CDCl3,
500 MHz): ꢀ 0.88 (t, J D6.8Hz, 6H), 1.25 (s, 44 H), 1.51–1.58 (m, 4 H), 2.35 (s, 6H, N–
CH3), 2.41–2.58 (m, 2H, N–CH2), 3.39–3.64 (m, 7H).
2.3.5. (R)-1,2-bis-Tetradecyloxy-3-O-benzylpropane (19)
To a stirred suspension of sodium hydride (76.4g, 1.91mol, 60% in oil) in anhy-
drous dimethylformamide (400 mL) under argon atmosphere at 0°C, a solution of
(R)-1-O-benzyl glycerol (18) (87g, 0.47mol) was added in dimethylformamide
(470mL) over a period of 1h, while maintaining the internal temperature below
20°C. After stirring at room temperature for 2h, tetradecyl bromide (530.2g,
1.91 mol) was added at 0 °C over a period of 2h. After complete addition, the reaction
mixture was stirred at room temperature for 2h and then temperature was gradually
increased to 60°C, and stirred for 14h. The reaction mixture was cooled to 0°C,
added few drops of ice water and diluted with water (3L) and added 50 mL conc.

K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
353
HCl solution. The aqueous layer was extracted with ethyl acetate (2£ 1 L). The
organic layer was washed with brine (500mL) and dried over sodium sulfate. The
organic layer was concentrated under reduced pressure and the crude product was
puriWed by column chromatography over a silica gel (230–400 mesh) eluting with
2–10% ethyl acetate in hexane to obtain (R)-1,2-bis-tetradecyloxy-3-O-benzylpro-
pane (19) (217 g, 79%) as colorless oil. TLC (SiO2) hexane/ethyl acetate (1:9)
1
Rf » 0.53. H NMR (CDCl3, 500MHz): ꢀ 0.88 (t, J D6.6 Hz, 6H), 1.25 (br s, 44H),
1.49–1.58 (m, 4H), 3.39–3.60 (m, 9H), 4.54 (s, 2H, OCH2Ph), 7.23–7.32 (m, 5H, Ph-H).
2.3.6. (S)-1,2-bis-Tetradecyloxypropan-3-ol (20)
(R)-1,2-Bis-tetradecyloxy-3-O-benzylpropane (19) (103 g, 0.17 mol) was dissolved
in ethyl acetate (350mL) and hydrogenated with 10% palladium on carbon (2.4g) for
4 h at 50 psi. After Wltration of the catalyst, the solution was evaporated under
reduced pressure. The residue was dissolved in hot hexane (700 mL) and kept at
¡20°C overnight. The separated solid was Wltered and dried to aVord (S)-1,2-bis-
tetradecyloxypropan-3-ol (20) (77 g, 89%) as a white solid. Mp 44–45 °C. TLC (SiO2)
hexane/ethyl acetate (1:9) Rf » 0.17. 1H NMR (CDCl3, 500MHz): ꢀ 0.88 (t, J D6.7Hz,
6H), 1.25 (br s, 44H), 1.51–1.57 (m, 4H), 2.22 (br s, 1H, OH), 3.40–3.73 (m, 9H).
2.3.7. (R)-2-[2-(2,3-bis-Tetradecyloxypropoxy)ethoxy]tetrahydro-2H-pyran (21)
To a stirred suspension of (S)-1,2-bis-(tetradecyloxy)-propane-3-ol (20) (135 g,
0.278mol) in anhydrous dimethylformamide (540mL) under argon atmosphere at
0 °C, sodium hydride (39g, 0.97 mol, 60% in oil) was added over a period of 1h main-
taining the temperature below 15 °C. After stirring at room temperature for 2h, 2-(2-
bromoethoxy)tetrahydro-2H-pyran (204g, 0.976mol) was added at 0°C over a
period of 3h maintaining the temperature below 10 °C. The reaction mixture was
stirred at room temperature for 48 h. The reaction mixture was cooled to 0 °C and ice
water was added slowly to quench excess sodium hydride. The reaction mixture was
concentrated under reduced pressure to remove most of the dimethylformamide and
the crude solution was diluted with water (3L) and extracted with ethyl acetate (1 L).
The organic layer was washed with aqueous saturated sodium chloride (500mL) and
dried over sodium sulfate. The solvent was concentrated under reduced pressure. The
crude product was puriWed by column chromatography over silica gel (230–400
mesh) with 1–8% ethyl acetate in hexane to obtain (R)-2-[2-(2,3-bis-tetradecyloxy-
propoxy)ethoxy]tetrahydro-2H-pyran (21) (138g, 81%) as colorless oil. TLC (SiO2)
hexane/ethyl acetate (1:4) Rf »0.30; 1H NMR (CDCl3, 500 MHz): ꢀ 0.88 (t,
J D6.7 Hz, 6H), 1.25 (s, 44H), 1.53–1.87 (m, 10H), 3.40–3.65 (m, 13H), 4.81–4.56 (m,
2H), 4.62–4.67 (m, 1H).
2.3.8. (R)-2-(2,3-bis-Tetradecyloxypropoxy)ethanol (22)
To a solution of (R)-2-[2-(2,3-bis-tetradecyloxypropoxy)ethoxy]tetrahydro-2H-
pyran (21) (105g, 0.17mol) in methanol (1 L) was added 1 M HCl in ether (5mL) and
stirred at room temperature for 2h. The reaction mixture was neutralized with solid
sodium bicarbonate until the solution became neutral. The reaction mixture was
Wltered and concentrated under reduced pressure. The residue was dissolved in ethyl

354
K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
acetate (1 L) and washed with water (200mL) and dried over sodium sulfate. The
organic layer was concentrated under reduced pressure and crude product was puri-
Wed by column chromatography over a silica gel (230–400 mesh) eluting with 5–15%
ethyl acetate in hexane to obtain (R)-2-(2,3-bis-tetradecyloxypropoxy)ethanol (22)
(72g, 79%) as colorless oil. TLC (SiO2) hexane/ethyl acetate (1:4) Rf »0.18. 1H NMR
(CDCl3, 500 MHz): ꢀ 0.88 (t, J D 6.7Hz, 6H), 1.25 (s, 44H), 1.51–161 (m, 4H), 2.58 (br
s, 1H, OH), 3.41–3.73 (m, 13H).
2.3.9. (R)-1-[1-(2-Bromoethoxymethyl)-2-tetradecyloxyethoxy]tetradecane (23)
To
a solution of (R)-2-(2,3-bis-tetradecyloxypropoxy)ethanol (22) (54 g,
102.1 mmol) in anhydrous dichloromethane (540mL) under argon atmosphere at
0 °C triphenylphosphine (34.7g, 132.7 mmol) was added followed by carbon
tetrabromide (43.9 g, 132.7 mmol). The reaction mixture was stirred at 0 °C for 2 h
and diluted with water (1.5L). The organic layer was separated, dried over sodium
sulfate, and concentrated under reduced pressure. The crude product was puriWed by
column chromatography over a silica gel (230–400 mesh) with 5% ethyl acetate in
hexane to obtain (R)-1-[1-(2-bromoethoxymethyl)-2-tetradecyloxyethoxy]tetrade-
cane (23) (57 g, 94%) as colorless oil. TLC (SiO2) hexane/ethyl acetate (1:5) Rf » 0.65.
1H NMR (CDCl3, 500 MHz): ꢀ 0.88 (t, J D6.7 Hz, 6H), 1.25 (s, 44H), 1.51–157 (m,
4H), 3.40–3.61 (m, 11H), 3.79 (t, J D 6 Hz, 2H).
2.3.10. (R)-[2-(2,3-bis-Tetradecyloxypropoxy)ethyl]dimethylamine (24)
(R)-1-[1-(2-Bromoethoxymethyl)-2-tetradecyloxyethoxy]tetradecane (23) (54 g,
91.3 mmol) was dissolved in 2 M methanolic solution of dimethylamine (810 mL) in
a screw-top pressure bottle. The pressure bottle was sealed and heated in an oil
bath while stirring at 88–90 °C for 60 h. The pressure bottle was cooled to room
temperature before it was opened. The solution was concentrated under reduced
pressure. The crude product was dissolved in ethyl acetate (1 L) and washed with
water (1 L). The organic layer was concentrated under reduced pressure and puri-
Wed by column chromatography over a silica gel (230–400 mesh) with 5–50% ethyl
acetate in hexane as eluent to obtain (R)-[2-(2,3-bis-tetradecyloxyprop-
oxy)ethyl]dimethylamine (24) (29 g, 57%) as light colored oil. TLC (SiO2) metha-
1
nol/chloroform (1:9) Rf » 0.46. H NMR (CDCl3, 500 MHz): ꢀ 0.88 (t, J D 6.7 Hz,
6H), 1.25 (s, 44H), 1.52–1.56 (m, 4H), 2.26 (s, 6H, N-CH3), 2.52 (t, J D 5.8 Hz, 2H),
3.39–4.61 (m, 11H). 13C NMR (CDCl3, 125 MHz): ꢀ 14.07, 22.65, 26.06, 26.11, 29.33,
29.47, 29.61, 29.63, 29.64, 29.66, 29.67, 30.08, 31.89, 45.91, 58.82, 69.79, 70.56, 71.29,
71.61, 77.83.
2.4. Synthesis of cationic cardiolipin analogues
2.4.1. 1,3-bis-(1,2-Ditetradecyloxypropyl-3-N,N-dimethyl ammonium bromide)
propane-2-ol (2) [PCL-1]
A solution of (R)-1,2-bis-tetradecyloxy-3-dimethylamino propane (17) (3.51 g,
6.88 mmol) and 1,3-dibromopropan-2-ol (8) (0.3 g, 1.37 mmol) in anhydrous
ethanol (30 mL) was reXuxed for a period of 7 days. The reaction mixture was

K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
355
cooled and the solvent was evaporated to give a crude waxy solid. The crude
compound was dissolved in hot hexane (50 mL) and stirred at room temperature
for 6 h. The separated solid was Wltered and washed with cold hexane (3 £ 10 mL)
to remove the starting material (R)-1,2-bis-tetradecyloxy-3-dimethylamino pro-
pane. The solid was dissolved in dichloromethane and acetone was added (ratio
1:10). The Xask was sealed and stored at 0 °C overnight. The white solid was
Wltered and washed with cold acetone (20 mL). The recrystallization procedure
was repeated two times. The compound was dried for 24 h under high vacuum
and then over P2O5 for 36 h to obtain cationic cardiolipin analogue (2) (1.3 g,
76%) as a white solid. Mp 106–108 °C. TLC (SiO2) methanol/chloroform (1:9) sin-
1
gle spot Rf » 0.11. H NMR (CDCl3, 500 MHz): ꢀ 0.88 (t, J D 6.8 Hz, 12H), 1.24–
1.31 (m, 88H), 1.53–1.57 (m, 8H), 2.08 (s, 1H, OH), 3.42–3.86 (m, 28H), 3.97 (d,
J D 13.5 Hz, 1H), 4.20 (t, J D 5.0 Hz, 1H), 4.37 (t, J D 4.5 Hz, 1H), 4.55 (t,
J D 12.5 Hz, 2H), 5.18–5.24 (m, 1H), 6.57 (d, J D 10.5 Hz, 1H). 13C NMR (CDCl3,
125 MHz): ꢀ 14.03, 22.60, 25.94, 25.96, 26.16, 29.28, 29.37, 29.39, 29.52, 29.55,
29.56, 29.58, 29.61, 29.63, 29.99, 30.00, 31.84, 52.78, 53.16, 54.89, 62.30, 64.99, 66.36,
68.52, 68.61, 68.94, 69.28, 69.40, 69.89, 72.01, 72.02, 72.80, 72.85; IR 3407 (br), 3223
(br), 2956 (s), 2920 (s), 1633, 1467, 1377, 1116, 971, 893, 720 cm¡1; ESI-MS 1159.4
[M+1-Br], 1079.9 [M+1-2Br], 542.2 [M+1–2Br/2]; Anal. Calcd for
C69H144Br2N2O5: C, 66.74; H, 11.69; N, 2.26; Br, 12.87. Found: C, 66.32; H, 11.62;
N, 2.31; Br, 13.27.
2.4.2. 1,3-bis-(1,2-Ditetradecyloxypropyl-3-N,N-dimethyl-3-ethoxy ammonium
bromide)propane-2-ol (3) [PCL-2]
A solution of (R)-1,2-bis-tetradecyloxy-3-dimethylamino propane (17) (35.6g,
69.8mmol) and 1,3-bis-(2-bromoethoxy)propane-2-ol (12) (7.1g, 23.2mmol) in anhy-
drous ethanol (430 mL) was reXuxed at 78–80 °C over a period of 5 days. The hot
reaction mixture was transferred to Erlenmeyer Xask and acetone (4.3 L) was added
dropwise while stirring over a period of 2h. The mixture was kept at ¡20 °C over-
night. The solid was Wltered and washed with cold acetone (500 mL) to obtain a col-
orless white solid (28g). The crude solid was puriWed by recrystallization in the
mixture of warm methanol and acetone (ratio 1:10) and then stored at 0 °C over-
night. The solid was separated, Wltered, and washed with cold acetone (300 mL). The
recrystallization was repeated two times to yield a pure product. The compound was
desiccated for 24h under high vacuum and then over P2O5 for 36 h to obtain cationic
cardiolipin analogue (3) (24 g, 78%) as a white solid. Mp 159–160°C; TLC (SiO2)
methanol/chloroform (1:9) single spot Rf »0.13. 1H NMR (CDCl3, 500 MHz): ꢀ 0.88
(t, J D 6.7Hz, 12H), 1.25 (s, 88H), 1.52–1.71 (m, 8H), 3.41–3.68 (m, 29H), 3.95–4.19 (m,
14H), 4.63 (br s, 1H, OH). 13C NMR (CDCl3, 125 MHz): ꢀ 13.88, 22.45, 25.84, 25.98,
29.14, 29.23, 29.27, 29.38, 29.44, 29.46, 29.49, 29.82, 31.70, 52.93, 53.00, 53.42, 64.95,
66.48, 68.63, 68.77, 69.12, 71.75, 72.58, 73.24; IR 3395 (br), 2917 (s), 2872 (s), 1467 (s),
1126 (br) cm¡1; ESI-MS 1248.5 [M+1-Br], 584.2 [M+1–2Br/2] Anal. Calcd for
C73H152Br2N2O7: C, 65.93; H, 11.52; N, 2.11; Br, 12.02. Found: C, 65.65; H, 11.49; N,
2.13; Br, 12.17.

356
K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
2.4.3. 1,3-bis-(1,2-Ditetradecyloxy-4-oxa-hexyl-6-N,N-dimethyl ammonium
bromide)propane-2-ol (4) [PCL-3]
A solution of (R)-[2-(2,3-bis-tetradecyloxypropoxy)ethyl]dimethylamine (24)
(39g, 70.2 mmol) and 1,3-dibromopropan-2-ol (8) (5.1 g, 23.4 mmol) in anhydrous
ethanol (440mL) was reXuxed at 78–80 °C over a period of 7 days. The reaction mix-
ture was cooled and the solvent was evaporated to give a crude waxy solid. The crude
compound was dissolved in hot hexane (200mL), stirred at room temperature for
60min, and kept at ¡20 °C for 2 h. The separated solid was Wltered and washed with
cold hexane (3£ 50 mL) to remove excess starting material (R)-[2-(2,3-bis-tetradecyl-
oxypropoxy)ethyl]dimethylamine. The solid was dissolved in warm methanol and
acetone (ratio 1:10). The Xask was stored at 0°C overnight. The white solid was
Wltered and washed with cold acetone (100mL). The recrystallization procedure was
repeated two times. The compound was desiccated for 24 h under high vacuum and
then over P2O5 for 36h to obtain cationic cardiolipin analogue (4) (23 g, 74%) as a
white solid. Mp 190–193 °C. TLC (SiO2) methanol/chloroform (1:9) single spot
1
Rf » 0.12. H NMR ꢀ (CDCl3, 500 MHz): ꢀ 0.88 (t, J D 6.8Hz, 12H), 1.25 (s, 88H),
1.49–1.58 (m, 8H), 3.39–3.72 (m, 32H), 3.81–4.01 (m, 8H), 4.49 (d, J D13.0 Hz, 2H),
5.24–5.30 (m, 1H), 6.48 (d, J D 9.0Hz, 1H). 13C NMR (CDCl3, 125MHz): ꢀ 13.98,
22.56, 25.97, 26.00, 29.23, 29.37, 29.38, 29.52, 29.54, 29.57, 29.58, 30.02, 31.79, 52.95,
52.98, 54.13, 61.71, 65.12, 65.66, 65.70, 66.56, 69.82, 69.83, 70.43, 70.44, 71.53, 71.56,
71.72, 77.37; IR 1117 (br s), 1467, 2917 (s), 2872 (s), 3398 (br s) cm¡1; ESI-MS: 1248.7
[M+1-Br¡], 584.6 [M+1–2Br¡/2]; Anal. Calcd for C73H152Br2N2O7: C, 65.93; H, 11.52;
N, 2.11; Br, 12.02. Found: C, 65.57; H, 11.36; N, 2.12; Br, 12.22.
2.4.4. 1,3-bis-(1,2-Ditetradecyloxy-4,10-dioxa-decyl-7-N,N-dimethyl ammonium
bromide)propane-2-ol (5) [PCL-4]
A solution of (R)-[2-(2,3-bis-tetradecyloxypropoxy)ethyl]dimethylamine (24)
(21g, 37.88 mmol) and 1,3-bis-(2-bromoethoxy)propane-2-ol (12) (3.84g, 12.6mmol)
in anhydrous ethanol (250mL) was reXuxed at 78–80 °C over a period of 7 days. The
hot reaction mixture was transferred to an Erlenmeyer Xask and acetone (2 L) was
added dropwise, while stirring over a period of 3h and kept at ¡20°C overnight. The
solid was Wltered and washed with cold acetone (100mL) to obtain a colorless white
solid (18g). The crude solid was puriWed by recrystallization in warm methanol and
acetone (ratio of 1:10) and then stored at ¡20°C overnight. The solid was separated,
Wltered, and washed with cold acetone (50 mL). The recrystallization was repeated
two times to yield pure compound. The compound was desiccated for 24h and then
over P2O5 for 36 h to obtain cationic cardiolipin analogue (5) (14g, 78%) as a white
solid. Mp 198–200 °C. TLC (SiO2) methanol/chloroform (1:9) single spot Rf » 0.13.
1H NMR (CDCl3, 500MHz): ꢀ 0.88 (t, J D 6.9Hz, 12H), 1.25 (s, 88H), 1.48–1.59 (m,
8H), 3.38–3.65 (m, 36H), 3.89–4.13 (m, 16H). 13C NMR (CDCl3, 125 MHz): 13.89,
22.46, 25.87, 25.90, 29.14, 29.28, 29.30, 29.43, 29.45, 29.48, 29.92, 31.70, 52.77, 52.83,
64.37, 64.87, 64.98, 65.36, 68.77, 69.99, 70.32, 71.35, 71.59, 72.62, 77.27. IR 1131, 1470,
2917, 2956, 3339cm¡1; ESI-MS: 1336.4 [M+1-Br¡], 628.4 [M+1–2Br¡/2]. Anal. Calcd
for C77H160Br2N2O9: C, 65.22; H, 11.37; N, 1.98; Br, 11.27. Found: C, 64.91; H, 11.27;
N, 1.98; Br, 11.18.

K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
357
2.5. Preparation of liposomes
Liposomes were prepared from newly synthesized cationic lipids and helper lipids
using thin-Wlm hydration method [23]. In brief, cationic lipid and DOPE were dis-
solved in chloroform in a round-bottomed Xask. The solvent was removed under
reduced pressure using rotary evaporator to form a lipid Wlm and further dried under
vacuum overnight. The lipid Wlm was hydrated with MilliQ water. The bulk cationic
liposomes were extruded through 0.2ꢀm pore size polycarbonate Wlters (Whatman)
three times and 0.1ꢀm pore size polycarbonate Wlters Wve times. The extruded lipo-
somes were sterilized by Wltering through 0.22 ꢀm sterile Wlter unit (Millipak 20 posi-
tively charged). The prepared liposomes had a mean particle size of <130nm. The
size of the liposomes was characterized using a light scattering particle sizer (Nicomp
Model 380, Santa Barbara, CA).
2.6. Cell culture and in vitro transfection
Chinese hampster ovary (CHO) cells were purchased from American Type Cul-
ture Collection (Manassas, VA) and were maintained in Kaighn’s modiWcation of
Ham’s F12 medium (F12K) with 10% HI-FBS. At one day prior to transfection,
CHO cells (10,000 cells per well) were seeded in 96-well plates and then cultured
overnight in a 5% CO2 incubator. The cells were then washed with phosphate-
buVered saline to remove the residual serum before adding the transfection
mixture.
The transfection eYciency of prepared liposomes was determined using ꢁ-galacto-
sidase (ꢁ-gal) reporter gene assay (Promega, Madison, WI) as previously described
[24]. The optical density of the sample was read at 414 nm using a plate reader
(Thermo Electron, Franklin, MA). The amount of ꢁ-gal expression was calculated
based on exogenous ꢁ-gal standard.
3. Results and discussions
The synthesis of bromo intermediates 8 and 12 is outlined in Scheme 1. Commer-
cially available 2-O-benzyl glycerol 6 was brominated with triphenylphosphine and
carbon tetrabromide in dichloromethane to aVord 1,3-dibromo-2-O-benzyl glycerol
7 in yield of 87%. Debenzylation of 7 via hydrogenation over 10% Pd/C catalyst in
ethanol gave 1,3-dibromopropan-2-ol 8 in 94% yield. Alkylation of 2-O-benzyl glyc-
erol 6 with 2-(2-bromoethoxy)tetrahydro-2H-pyran in the presence of sodium
hydride (60% in oil) in dimethylformamide aVorded 1,3-bis-[(2-ethoxy tetrahydro-
2H-pyran)]-2-O-benzyl glycerol 9 in 71% yield. The THP derivative 9 on deprotec-
tion in methanol with catalytic amount of 1M HCl solution in diethyl ether at room
temperature for 2 h gave the corresponding diol derivative 3,7-dioxa-5-O-benzyl-1,9-
nonanediol 10 in 88% yield. The diol 10 on bromination using Ph3P/CBr4 in CH2Cl2
yielded 1,9-dibromo-3,7-dioxa-5-O-benzyl nonane 11 in 91% yield. Debenzylation of
11 via hydrogenation provided 1,3-bis-(2-bromoethoxy)propane-2-ol 12 in 94% yield.

358
K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
OBn
OBn
7
OH
8
a
b
HO
OH
Br
Br
Br
Br
6
c
OBn
10
OBn
9
a
d
O
O
O
O
HO
OH
THPO
OTHP
OH
12
OBn
b
O
O
O
O
Br
Br
Br
Br
11
Scheme 1. Preparation of bromo intermediate. (a) Ph3P, CBr4, 0 °C, (b) 10% Pd-C, H2, 50 psi, ethanol, (c)
NaH, BrCH2CH2OTHP, DMF, 0 °C to rt, (d) 1 M HCl in diethyl ether, MeOH, rt.
The synthesis of tertiary amine intermediates 17 and 24 is outlined in Scheme 2.
Williamson etheriWcation of commercially available (S)-1-O-benzyl glycerol 13
with tetradecyl bromide in the presence of sodium hydride (60% in oil) in dimethyl-
formamide aVorded (S)-1,2-bis-tetradecyloxy-3-O-benzylpropane 14 in 75% yield.
Debenzylation of 14 via hydrogenation over 10% Pd/C catalysts in ethyl acetate
gave (R)-1,2-bis-tetradecyloxy propane-3-ol 15 in 92% yield. The alcohol 15 on
brominating using Ph3P/CBr4 in dichloromethane aVorded (S)-1,2-bis-tetradecyl-
oxy-3-bromopropane 16 in 90% yield. The bromo compound 16 on heating with
10-fold excess of 2 M methanolic dimethylamine solution in pressure bottle gave
(R)-1,2-bis-tetradecyloxy-3-N,N dimethylamine propane 17 (88% yield). Similarly,
commercially available (R)-1-O-benzyl glycerol 18 was converted to (S)-1,2-bis-
tetradecyloxy propane-3-ol 20 in two steps. Alkylation of (R)-1-O-benzyl glycerol
18 with tetradecyl bromide in the presence of sodium hydride (60% in oil) in
dimethylformamide aVorded (S)-1,2-bis-tetradecyloxy-3-O-benzylpropane 19 in
79% yield. Debenzylation of 19 via hydrogenation over 10% Pd/C catalysts in ethyl
acetate gave (S)-1,2-bis-tetradecyloxy propane-3-ol 20 in 89% yield. Alkylation of
20 with 2-(2-bromoethoxy)tetrahydro-2H-pyran in the presence of sodium hydride
in dimethylformamide aVorded (R)-2-[2-(2,3-bis-tetradecyloxypropoxy)eth-
oxy]tetrahydropyran 21 in 81% yield. The THP derivative 21 was deprotected in
methanol with catalytic amount of 1 M HCl solution in diethyl ether at room tem-
perature for 2 h to yield 79% of the corresponding alcohol, (R)-2-(2,3-bis-tetrade-
cyloxypropoxy)ethanol 22. The alcohol 22 on bromination using Ph3P/CBr4 in
dichloromethane yielded 94% of (R)-1-[1-(2-bromoethoxymethyl)-2-tetradecyloxy-
ethoxy]tetradecane 23. The bromo compound 23 on heating with 10-fold excess of
2 M methanolic dimethylamine solution in pressure bottle gave (R)-[2-(2,3-bis-
tetradecyloxypropoxy)ethyl]dimethylamine 24 in 57% yield.

K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
359
HO
HO
C
C
H
O
O
C
C
H
O
O
c
a
14 29
14 29
b
H
H
14 29
14 29
OBn
OH
OBn
13
(S)-1-O-benzyl glycerol
15
14
C
C
H
O
O
14 29
d
C
C
H
O
O
14 29
H
14 29
H
14 29
Br
N
16
17
C
C
H
O
O
C
C
H
O
O
14 29
e
HO
HO
a
14 29
b
H
14 29
H
14 29
OH
OBn
OBn
19
20
18
(R)-1-O-benzyl glycerol
C
C
H
O
O
14 29
C
H O
14 29
c
f
H
14 29
C
14 29
H
O
OTHP
OH
O
O
21
22
C
H
O
O
14 29
C
H O
14 29
d
C
14 29
H
C
H
O
14 29
Br
N
O
O
23
24
Scheme 2. Preparation of tertiary amine intermediate. (a) NaH, C14H29Br, DMF, 60–70 °C, (b) 10% Pd-C,
H2, 50 psi, ethyl acetate, (c) Ph3P, CBr4, CH2Cl2, 0 °C, (d) 2 M dimethylamine in methanol, pressure bottle,
90 °C, (e) NaH, BrCH2CH2CH2OTHP, DMF, 0 °C to rt, (f) 1 M HCl in diethyl ether, MeOH, rt.
The synthesis of cationic cardiolipin analogues 2, 3, 4, and 5 is outlined in Scheme
3. The bromo compounds 8 and 12 were reacted with 17 in ethanol at reXux
temperature for 5–7 days to yield cationic cardiolipin analogues 1,3-bis-(1,2-dite-
tradecyloxypropyl-3-N,N-dimethyl ammonium bromide)propane-2-ol 2 (76%) and
1,3-bis-(1,2-ditetradecyloxypropyl-3-N,N-dimethyl-3-ethoxy ammonium bromide)
propane-2-ol 3 (78%), respectively. The bromo compounds 8 and 12 were reacted
with 24 in ethanol under reXux temperature for 5–7 days to yield cationic cardiolipin
analogues, 1,3-bis-(1,2-ditetradecyloxy-4-oxa-hexyl-6-N,N-dimethyl ammonium

360
K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
C14H29
O
O
C14H29
3 (PCL-2)
5 (PCL-4)
OH
8
(PCL-1) 2
OH
12
N
Br
Br
O
O
17
Br
N
Br
Ethanol, reflux
Ethanol, reflux
C14H29
C14H29
O
(PCL-3) 4
O
O
24
Scheme 3. Synthesis of cationic cardiolipin analogues.
bromide)propane-2-ol 4 (74%) and 1,3-bis-(1,2-ditetradecyloxy-4,10-dioxa-decyl-7-
N,N-dimethyl ammonium bromide)propane-2-ol 5 (78%), respectively.
The transfection eYciencies of new cationic lipids 4 (PCL3) and 5 (PCL4) were
evaluated and compared with PCL2-based transfection reagent, NeoPhectin at a
charge ratio of 1:1 cationic lipid/DNA (+/¡) (Fig. 5). NeoPhectin is a currently mar-
keted transfection reagent composed of cationic lipid 3 (PCL2): DOPE at a molar
ratio of 1:2 and showed high transfection among the cell lines including CHO cells
[24]. Further, liposome containing compound 3 showed lower toxicity in mice than
the DOTAP-based In Vivo GeneSHUTTLE [24]. In this study, the new cationic lip-
ids (4 and 5) were formulated having cationic lipid:DOPE ratio 1:2 similar to NeoP-
hectin. The results showed that the transfection eYciency of formulation containing
compound 4 is similar to NeoPhectin while compound 5 exhibit lower transfection.
The presence of additional two oxyethylene groups in compound 5 may have resulted
in a shape other than cone and hence showed low transfection. We anticipate cationic
lipids 4 and 5 to exhibit lower toxicity similar to compound 3.
Transfection efficiency of cationic lipids
2.50
2.00
1.50
1.00
0.50
0.00
Fig. 5. Transfection of liposomes containing cationic lipids 3, 4, 5 in vitro. The experiment was repeated
four times with triplicate wells for each set.

K. Kasireddy et al. / Bioorganic Chemistry 33 (2005) 345–362
361
4. Conclusion
We have prepared a series of new cationic cardiolipin analogues. The process is
scalable from gram to kilogram scale. Formulations containing 3 (PCL-2, NeoPhec-
tin) have been shown to transfect [24,25] diVerent cell lines with up to Wve times more
eYciency compared to commonly used Lipofectin. Formulations containing com-
pound 5 exhibited similar transfection properties when compared to NeoPhectin.
These cationic cardiolipin analogues could be used to formulate a broad range of
therapeutic agents, including antisense oligonucleotide as well as gene transfection
agents. Application of these cationic cardiolipin analogues to deliver antisense oligo-
nucleotide (AON) [26,27] and siRNA [28] into the targeted cells in vitro and in vivo is
under investigation and will be published in future.
Acknowledgment
We thank the Bioanalytical group of Pharmacokinetics, Safety, and EYcacy (PSE)
Department at NeoPharm for the Mass spectral analyses.
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