Synthesis of novel dimeric cationic lipids based on an aromatic backbone between the hydrocarbon chains and headgroup

Article abstract of DOI:10.1016/j.tetlet.2006.09.072

Seven dimeric cationic lipids possessing an aromatic anchor between the hydrocarbon chains and cationic headgroup have been synthesized. The spacers in these lipids vary in length, hydrophobicity and flexibility. The synthesis, membrane-forming properties

Full text of DOI:10.1016/j.tetlet.2006.09.072

Tetrahedron Letters 47 (2006) 8401–8405
Synthesis of novel dimeric cationic lipids based on an
aromatic backbone between the hydrocarbon chains and headgroup
Bishwajit Paul,a Avinash Bajaj,a S. S. Indib and Santanu Bhattacharyaa,*
aDepartment of Organic Chemistry, Indian Institutue of Science, Bangalore 560 012, India
bDepartment of Microbiology and Cell Biology, Indian Institutue of Science, Bangalore 560 012, India
Received 14 June 2006; revised 6 September 2006; accepted 14 September 2006
Available online 10 October 2006
Abstract—Seven dimeric cationic lipids possessing an aromatic anchor between the hydrocarbon chains and cationic headgroup
have been synthesized. The spacers in these lipids vary in length, hydrophobicity and flexibility. The synthesis, membrane-forming
properties and complexation with plasmid DNA (lipoplex formation) are briefly described.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The use of genes as therapeutic materials holds much
promise for practical clinical applications. Indeed, many
clinical trials in this field are currently in progress.1
Towards this end, engineered viruses have been shown
to be highly efficient vectors,2 but a number of severe
setbacks have recently imposed serious limits on their
medical use.3 This has intensified the search for syn-
thetic vectors such as cationic lipids4 which do not elicit
immunogenic reactions when administered.
Cationic lipid suspensions readily form complexes with
negatively charged DNA (the gene) under ambient
conditions. The molecular structure of cationic lipids is
an important parameter that controls their DNA
+
+
+
SPACER
N
N
N
RO
OR
z)-C18H35
RO
OR
RO
OR
R = 9(
Dimeric Lipids
DOTMA
(I)
spacer = hydrophobic or hydrophilic
-C(O)-9(z)-C17H33
R =
DOTAP
(II)
+
+
+
SPACER
N
N
NMe3
H33C16O
OC16H33
OC16H33
OC16H33
OC16H33
OC16H33
2, 3
1
Dimeric Lipids
spacer = Flexible or rigid
Figure 1. Schematic of the Gemini lipids based on glycerol or aromatic backbone. Transfection active lipids, for example, DOTMA (I) and DOTAP
(II) and monomeric counterpart of the aromatic gemini are also shown.
Keywords: Dimeric lipids; Vesicles; Gene transfer.
*
Corresponding author. Tel.: +91 80 22932664; e-mail: sb@orgchem.iisc.ernet.in
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.09.072

8402
B. Paul et al. / Tetrahedron Letters 47 (2006) 8401–8405
complexation and gene transfection activity. For
instance, the functional group that links the polar head
group and the hydrocarbon chains of such lipid mole-
cules plays a crucial role in their utilization in gene
transfer events. Thus, N-[1-(2,3-dioleyloxy)-N,N,N-
trimethylammonium chloride (DOTMA) (I), which
contains an ether linkage between the head group and
the long alkyl chains shows greater in vivo transfection
efficiency than the corresponding lipid possessing
an ester N-[1-(2,3-dioleyl)-N,N,N-trimethylammonium
chloride (DOTAP) (II),5 (Fig. 1).
the presence of the aromatic ring and straight hydrocar-
bon chain of a length depending on the source of such
lipids.8
For the last few years, we have been examining the role
of various molecular level modifications on the mem-
brane forming properties of different lipids.9,10 Most of
these lipid designs included molecules based on natural
glycerol based architectures. Herein, we describe a con-
venient synthesis of the above type of cationic lipids 1–3
(Fig. 1) and briefly report their membrane forming and
DNA binding properties.
Recently, another class of lipids (in the dimeric form)
known as dimeric lipids, have also received attention
due to their better gene transfection abilities compared
to their monomeric counterparts.6 Clearly the design
and syntheses of new lipid systems with alternative
structural types are crucial for the development of
potent synthetic vectors for such applications.
The synthesis of the key precursor molecule 7 for all the
lipids is outlined in Scheme 1. Firstly the –CO2H moiety
of 3,4-dihydroxybenzoic acid was protected via esterifi-
cation in the presence of concd. HCl and dry MeOH
under reflux conditions to afford the corresponding ester
4 in 90% yield. The hydroxyl groups of ester 4 were then
subjected to alkylation with n-hexadecyl bromide by re-
flux in acetone in the presence of K2CO3 to furnish the
dialkylated product 5 in 77% yield. The product 5 was
then reduced with LiAlH4 in dry THF, under reflux over
a period of 12 h, to afford 6 in 97% isolated yield. Next,
the conversion of 6 to the corresponding benzylic bro-
mide was attempted. However, under Appel conditions
(CBr4/PPh3 in CH2Cl2), although TLC indicated satis-
factory conversion, during the isolation, the product
decomposed. The conversion of the alcohol to the corre-
sponding bromide by PBr3 in CH2Cl2, appeared to be
complete as judged by TLC. However, again it was
not possible to isolate the corresponding bromide pre-
sumably because of its high instability. Therefore upon
confirming the formation of the bromide after reaction
with PBr3 in CH2Cl2 for 24 h by TLC, a methanolic
solution of dimethylamine was introduced directly into
the reaction mixture and the mixture was stirred at room
temperature for 12 h. After work-up, compound 7 was
obtained in 86% yield.
In search of new targets for lipid design, we envisioned
developing dimeric lipids based on aromatic back-
bones.7 We thought that it would be of interest to design
and synthesize lipids based on such structural anchors
and investigate their membrane-forming properties.
In particular, the presence of sp2 hybridized planar
aromatic hydrocarbon rings at the hydrocarbon
chain-polar headgroup linkage region pose interesting
situations pertaining to their inter-lipidic interactions
in membranes as opposed to those based on pseudogly-
ceryl backbones, which associate with each other and
the interfacial water via hydrogen bonding and dipolar
interactions.
Lipids based on aromatic backbones are rare in eukaryo-
tic organisms, although their presence has been demon-
strated in marine sponges.8 Among aromatic ring
compounds, those considered as lipids are, however,
formed in some plants and contain a catechol, resorcinol
or hydroquinone nucleus alkylated with a long hydro-
carbon chain. Such resorcinolic lipid molecules have a
dual, aromatic and acyclic character, demonstrated by
The lipid 1 was prepared upon refluxing the tertiary
amine 7 with an excess of MeI in EtOH in quantitative
CH2OH
COOMe
COOMe
COOH
(iii)
(ii)
(i)
OC16H33
OC16H33
OH
OH
OC16H33
OC16H33
OH
OH
6
4
5
+
NMe
-
I
NMe2
,
3
(v)
(iv)
6
OC16H33
OC16H33
OC16H33
OC16H33
1
7
Scheme 1. Reagents, conditions and yields: (i) HCl, MeOH, 80 ꢁC, 12 h, 90%; (ii) n-C16H33Br, K2CO3, dry acetone, 65 ꢁC, 84 h, 77%; (iii) LiAlH4,
dry THF, 70 ꢁC, 12 h, 97%; (iv) (a) PBr3, CH2Cl2, rt, 24 h; (b) Me2NH (excess), MeOH, rt, 12 h (overall yield = 86% for steps (a) and (b)) and (v)
MeI, EtOH, 80 ꢁC, 24 h, screw-top pressure tube, quantitative yield.

B. Paul et al. / Tetrahedron Letters 47 (2006) 8401–8405
8403
+
N
Br-
+
Br-
N
+
N
+
Br-
(ii)
Br-
N
(i)
7
H33C16O
OC16H33
OC16H33
H33C16O
OC16H33
OC16H33
OC16H33
(iii)
OC16H33
3a
3b
+
N
+
Br-
Br-
N
(CH2)n
a: n = 3
b: n = 4
c: n = 5
d: n = 6
H33C16O
OC16H33
OC16H33
OC16H33
e: n = 12
2
Scheme 2. Reagents, conditions and yields (i) a,a–dibromo-p-xylene, MeOH–EtOAc (2:1), 80 ꢁC, screw-top pressure tube, 24 h, 60%; (ii) a,a-
dibromo-m-xylene, MeOH–EtOAc (2:1), 80 ꢁC, screw-top pressure tube, 24 h, 60% and (iii) Br(CH2)nBr, MeOH–EtOAc (2:1), 80 ꢁC, screw-top
pressure tube, 2–4 days, 30–40%.
yield (Scheme 1). The dimeric cationic lipids with
polymethylene spacers, 2a–e were synthesized by
heating 7 with the appropriate a,x-dibromo alkanes
to 80 ꢁC in a mixture of MeOH–EtOAc for 2–4 days
in a screw-top pressure tube (Scheme 2). After repeated
crystallizations from a mixture of MeOH–EtOAc, the
isolated yields of the dimeric lipids ranged from 30%
to 40%. Compound 7 on reaction with a,a–dibromo-
p-xylene in a mixture of MeOH–EtOAc afforded the
dimeric lipid 3a with a hydrophobic rigid spacer
(Scheme 2). Similarly, the reaction of 7 with a,a-di-
bromo-m-xylene afforded the dimeric lipid 3b in 60%
yield (Scheme 2). Lipids 3a and 3b are of interest as
cyclophane macrocycles built with para- and meta-
xylenediamine.11
Sonication (15 min) of each of the newly synthesized lip-
ids above 70–80 ꢁC with an aqueous buffer afforded sta-
ble translucent suspensions. The existence of vesicle-like
organizations in these suspensions was evident from
transmission electron microscopy (Fig. 2). Gel retarda-
tion studies with the plasmid EGFP-c3 at different N/
P ratios, showed the retardation of the DNA-plasmid
at an N/P ratio of 1.0–2.0 for all the lipids (Fig. 3).
In summary, we have synthesized a series of novel cat-
ionic dimeric lipids, which will help in understanding
the effect of modulation of different spacers between
the headgroups in membranes. The method developed
is simple and efficient for the synthesis of a series of cat-
ionic dimeric lipids based on an aromatic backbone. The
process is scalable from the gram to kilogram scale.
These cationic dimeric lipids will be useful for the prep-
aration of a range of therapeutic formulations, including
gene transfection agents as well as for antisense DNA
technology. The application of these cationic liposomes
to deliver genetic materials into targeted cells in vitro
All the new lipids were purified by repeated crystalliza-
tions from MeOH–EtOAc and isolated as white solids.
All the numbered intermediates and the final prod-
1
ucts were characterized from their IR, H NMR, 13C
NMR and mass spectra.12
Figure 3. Gel retardation assay of dimeric lipid 2c at different N/P
ratios. (Lane 1: Plasmid alone, Lane 2: N/P = 0.5, Lane 3: N/P = 1.0,
Lane 4: N/P = 2.0, Lane 5: N/P = 4.0.)
Figure 2. Transmission electron micrograph of an aqueous suspension
of the dimeric lipid 2d showing the vesicular nature of the dimeric lipid.

8404
B. Paul et al. / Tetrahedron Letters 47 (2006) 8401–8405
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and in vivo is under investigation and will be reported in
due course.
Acknowledgements
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We thank the Department of Biotechnology, Govern-
ment of India, for support of this work and CSIR,
New Delhi, for the award of a Research Fellowship to
A.B. We also thank the Molecular Biophysics Unit,
IISc, for giving access to their mass spectral facility.
11. (a) Rastogi, R. P.; Khanna, N. M.; Dhar, M. L. J. Sci.
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References and notes
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12. All new compounds exhibited spectral characteristics
consistent with their given structures. Selected spectral
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3. Marshall, E. Science 2002, 298, 510–511.
1
data for the final compounds are as follows: Lipid 1. H
NMR (300 MHz, CDCl3) d: 0.85 (t, 6H, J = 7.0 Hz, 2 ·
–CH3), 1.25–1.46 (m, 52H, 26 · –CH2), 1.78–1.80 (m, 4H,
2 · –O–CH2–CH2–), 3.35 (s, 9H, 3 · –N+–CH3), 3.97–4.05
(m, 4H, –O–CH2–CH2–), 4.91 (s, 2H, –N+–CH2–Ar), 6.87
(d, 1H, J = 8.1 Hz, ArH), 7.11 (d, 1H, J = 8.1 Hz, ArH),
7.21 (s, 1H, ArH). 13C NMR (75 MHz, CDCl3) d: 14.10,
22.67, 26.02, 26.11, 29.15, 29.35, 29.43, 29.51, 29.71, 31.91,
52.77, 69.11, 69.86, 113.08, 117.93, 119.01, 126.10, 149.27,
151.27. ESIMS: 631.8 (M+). Lipid 2a. 1H NMR
(300 MHz, CDCl3) d: 0.87 (t, 12H, J = 7.0 Hz, 4 ·
–CH3), 1.25–1.46 (m, 104H, 52 · –CH2), 1.82–1.86 (m,
8H, 4 · –O–CH2–CH2–), 3.01–3.10 (bm, 2H, –N+–CH2–
CH2–), 3.21 (s, 12H, 4 · –N+–CH3), 3.96–4.01 (m, 12H,
–O–CH2–CH2– and –N+–CH2–CH2), 4.72 (s, 4H, 2 ·
–N+–CH2–Ar), 6.85–6.88 (d, 2H, J = 8.1 Hz, ArH), 7.07–
7.10 (m, 4H, ArH). 13C NMR (75 MHz, CDCl3 +
CD3OD) d: 13.87, 22.49, 25.91, 28.97, 29.16, 29.26,
29.35, 29.53, 31.73, 49.43, 61.80, 68.43, 68.96, 69.65,
113.04, 118.06, 118.62, 126.20, 149.05, 151.40. ESIMS:
4. For recent examples of cationic lipid design, see: (a)
Ahmed, O. A. A.; Pourzand, C.; Blagbrough, I. S. Pharm.
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1
701.7 (M+ÀCH2Ar(OC16H33)), 571.7. Lipid 2b. H NMR
(300 MHz, CDCl3) d: 0.87 (t, 12H, J = 7.0 Hz, 4 · –CH3),
1.25–1.46 (m, 104H, 52 · –CH2), 1.76–1.86 (m, 8H, 4 ·
–O–CH2–CH2–), 2.29–3.10 (m, 4H, 2 · –N+–CH2–CH2–),
3.12 (s, 12H, 4 · –N+–CH3), 3.99 (t, 8H, J = 6.0 Hz, 4 ·
–O–CH2–CH2–), 4.02–4.11 (m, 4H, –N+–CH2–CH2), 4.62
(s, 4H, 2 · –N+–CH2–Ar), 6.88 (d, 2H, J = 8.1 Hz, ArH),
7.05–7.12 (m, 4H, ArH). 13C NMR (75 MHz, CDCl3) d:
14.06, 26.06, 26.12, 29.20, 29.33, 29.48, 29.56, 29.71, 31.89,
49.33, 64.12, 68.05, 69.04, 69.81, 113.09, 118.12, 118.88,
126.37, 149.17, 151.16. ESIMS: 644.2 (M+2/2), 715.8.
Lipid 2c. 1H NMR (300 MHz, CDCl3) d: 0.88 (t, 12H,
J = 7.0 Hz, 4 · –CH3), 1.25–1.47 (m, 106H, 52 · –CH2
and –N+–CH2–CH2–CH2–), 1.79–1.85 (m, 8H, 4 · –O–
CH2–CH2–), 2.10–2.24 (bm, 4H, 2 · –N+–CH2–CH2–),
3.18 (s, 12H, 4 · –N+–CH3), 3.99–4.01 (m, 12H, 4 · –O–
CH2–CH2– and 2 · –N+–CH2–CH2), 4.75 (s, 4H, 2 ·
–N+–CH2–Ar), 6.87 (d, 2H, J = 8.1 Hz, ArH), 7.13 (m,
4H, ArH). 13C NMR (75 MHz, CDCl3) d: 14.03, 22.60,
26.07, 29.30, 29.43, 29.66, 31.85, 49.20, 64.25, 67.51, 68.97,
69.73, 112.98, 118.17, 119.18, 126.35, 149.05, 150.99.
6. (a) Chie, Pei-Yu; Wang, J.; Carbonaro, D.; Lei, S.; Bruce,
M.; Sheikh, S.; Ali, S. M.; Ahmad, M. U.; Ahmad, I.
Cancer Gene Ther. 2005, 12, 321–328; (b) Bell, P. C.;
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Wong, T.; Wong, K. F.; Maurer, N.; Cullis, P. R.
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Remy, J. S.; Blessing, T.; Behr, J. P. J. Am. Chem. Soc.
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Angew. Chem., Int. Ed. 2000, 39, 1907–1920; (f) Bhattach-
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2287–2288; (g) Menger, F. M.; Littau, C. A. J. Am. Chem.
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7. (a) Ewert, K. K.; Evans, H. M.; Zidovska, A.; Bouxsein,
N. F.; Ahmad, A.; Safinya, C. R. J. Am. Chem. Soc. 2006,
128, 3998–4006; (b) Koiwai, K.; Tokuhisa, K.; Karinaga,
R.; Kudo, Y.; Kusuki, S.; Takeda, Y.; Sakurai, K.
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Ahmad, A.; Evans, H. M.; Schmidt, H. W.; Safinya, C. R.
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1
ESIMS: 731.2, 650.2 (M+2/2), 571.7, Lipid 2d. H NMR
(300 MHz, CDCl3) d: 0.88 (t, 12H, J = 7.0 Hz, 4 ·
–CH3), 1.25–1.46 (m, 108H, 52 · –CH2 and 2 · –N+
–CH2–CH2–CH2–), 1.78–1.82 (m, 8H, 4 · –O–CH2
–CH2–), 2.24–2.40 (m, 4H, 2 · –N+–CH2–CH2–), 3.21 (s,
12H, 4 · –N+–CH3), 3.84–3.95 (m, 4H, –N+–CH2–CH2),
3.97–4.03 (m, 8H, 4 · –O–CH2–CH2–), 4.62 (s, 4H, –N+
8. Kozubek, A.; Tyman, J. H. P. Chem. Rev. 1999, 99, 1–26.
9. (a) Bhattacharya, S.; Subramanian, M. Tetrahedron Lett.
2002, 43, 4203–4206; (b) Bhattacharya, S.; Acharya, S. N.
G. Langmuir 2000, 16, 87–97; (c) Bhattacharya, S.; De, S.
Chem. Eur. J. 1999, 5, 2335–2347; (d) Bhattacharya, S.;
Dileep, P. V. Tetrahedron Lett. 1999, 40, 8167–8171;

B. Paul et al. / Tetrahedron Letters 47 (2006) 8401–8405
8405
–CH2–Ar), 6.87 (d, 2H, J = 8.1 Hz, ArH), 7.13–7.19 (m,
4H, ArH). 13C NMR (75 MHz, CDCl3) d: 14.06, 21.86,
22.63, 24.67, 26.02, 26.09, 29.16, 29.31, 29.43, 29.51, 29.62,
31.88, 49.51, 64.56, 67.62, 69.04, 69.76, 113.07, 118.22,
119.24, 126.33, 149.12, 151.06. ESIMS: 657.2 (M+2/2),
616.5, 571.3. Lipid 2e. 1H NMR (300 MHz, CDCl3) d:
0.86 (t, 12H, J = 7.0 Hz, 4 · –CH3), 1.16–1.40 (m, 124H,
3.99–4.13 (m, 8H, 4 · –O–CH2–CH2–), 4.87 (s, 4H, –N+
–CH2–Ar), 5.33 (s, 4H, –N+–CH2–Ar), 6.87 (d, 2H,
J = 8.1, ArH), 7.21–7.25 (m, 4H, ArH), 7.81 (s, 4H,
–CH2–C6H6–CH2–). 13C NMR (75 MHz, CDCl3) d: 14.20,
22.88, 26.29, 26.34, 29.38, 29.56, 29.66, 29.72, 29.87, 29.92,
32.13, 48–49 (mixed with CD3OD), 66.67, 68.23, 69.42,
70.13 113.52, 118.57, 119.05, 126.73, 130.18, 134.36,
149.54, 151.63. ESIMS: 763.5, 668.5 (M+2/2), 616.7,
52 · –CH2
and
2 · –N+–CH2–CH2–CH2–CH2–CH2
1
–CH2–), 1.76–1.78 (m, 8H, 4 · –O–CH2–CH2–), 3.17 (s,
12H, 4 · –N+–CH3), 3.62–3.70 (m, 4H, 2 · –N+–CH2
–CH2), 3.94–3.99 (m, 8H, 4 · –O–CH2–CH2–), 4.79 (s,
4H, –N+–CH2–Ar), 6.82 (d, 2H, J = 8.1 Hz, ArH), 7.09
(d, 2H, J = 8.1 Hz, ArH), 7.15–7.21 (m, 2H, ArH). 13C
NMR (75 MHz, CDCl3) d: 14.05, 22.62, 26.04, 26.07,
28.65, 29.15, 29.31, 29.41, 29.48, 29.67, 31.86, 49.36, 64.04,
67.62, 69.01, 69.70, 112.98, 118.14, 119.14, 126.27, 149.10,
151.04. ESIMS: 700.3 (M+2/2). Lipid 3a. 1H NMR
(300 MHz, CDCl3) d: 0.88 (t, 12H, J = 7.0 Hz, 4 ·
–CH3), 1.25–1.46 (m, 104H, 52 · –CH2), 1.78–1.90 (m,
8H, 4 · –O–CH2–CH2–), 3.13 (s, 12H, 4 · –N+–CH3),
571.3. Lipid 3b. H NMR (300 MHz, CDCl3) d: 0.88 (t,
12H, J = 7.0 Hz, 4 · –CH3), 1.25–1.46 (m, 104H, 52 ·
–CH2), 1.78–1.85 (m, 8H, 4 · –O–CH2–CH2–), 3.11 (s,
12H, 4 · –N+–CH3), 3.98–4.00 (m, 8H, 4 · –O–CH2–
CH2–), 4.84 (s, 4H, –N+–CH2–Ar), 5.14 (s, 4H, –N+
–CH2–Ar), 6.85 (d, 2H, J = 8.1 Hz, ArH), 7.13–7.24 (m,
4H, ArH), 7.27 (m, 1H, ArH), 7.40–7.48 (m, 1H, ArH),
7.79–7.84 (m, 2H, ArH). 13C NMR (75 MHz, CDCl3) d:
14.05, 22.65, 26.04, 26.10, 29.18, 29.31, 29.46, 29.53, 29.69,
31.86, 48.48, 66.67, 68.23, 68.99, 69.73, 112.98, 118.14,
118.90, 126.51, 126.80, 129.95, 135.25, 138.42, 149.15,
151.11. ESIMS: 763.3, 668.5 (M+2/2), 571.7.