A highly efficient approach for the synthesis of cationic lipid DOSPA

Article abstract of DOI:10.1055/s-2006-949636

A highly efficient strategy has been developed for the synthesis of the cationic lipid, N-[2-({2,5-bis[(3-aminopropyl)amino]-1-oxopentyl}amino)ethyl]-N, N-dimethyl-2,3-bis[(1-oxo-9-octadecenyl)oxy] salt with hydrogen chloride (DOSPA). It involves the linkage of a cationic head group and a hydrophobic moiety in the presence of the standard coupling reagents. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2006-949636

2284
LETTER
A Highly Efficient Approach for the Synthesis of Cationic Lipid DOSPA
S
ynthesis of Catio
a
nic Lipid D
n
OSPA hong Li,a Robert J. Debs,b Timothy D. Heath*a
a
School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, Wisconsin, 53705-2221, USA
Fax +1(608)2625345; E-mail: tdheath@pharmacy.wisc.edu
b
California Pacific Medical Center Research Institute, 475 Brannan St, Suite 220, San Francisco, CA 94107, USA
Received 23 February 2006
O
OR
Abstract: A highly efficient strategy has been developed for the
synthesis of the cationic lipid, N-[2-({2,5-bis[(3-aminopropyl)ami-
no]-1-oxopentyl}amino)ethyl]-N,N-dimethyl-2,3-bis[(1-oxo-9-oc-
tadecenyl)oxy] salt with hydrogen chloride (DOSPA). It involves
the linkage of a cationic head group and a hydrophobic moiety in the
presence of the standard coupling reagents.
+
N
OR
+
H3N
N
N
+
H2
H
+
NH2
R =
8
Key words: cationic lipids, DNA, synthesis, liposome, transfection
7
+
NH3
Figure 1 Structure of 2,3-dioleoyloxy-N-[2-(spermine-carboxami-
do)ethyl]-N,N-dimethyl-1-propanaminum (DOSPA).
In the past decade, the near completion of the human
genome sequencing project offers an unparalleled oppor-
tunity to understand the genetic basis of various disease1,
and makes gene therapy an excellent opportunity to cure
cancer or inherited diseases in the future.2–4 However,
gene therapy still faces significant technological hurdles
before it becomes an established therapeutic strategy.5–8
Development is required for systems that can efficiently
deliver the therapeutic gene or nucleic acid to the site of
disease while minimizing delivery to other sites.9
devise an efficient synthetic procedure for the multivalent
cationic lipid DOSPA, which we here report.
DOSPA is a typical cytofectin and contains a cationic
head group attached by a linker to a hydrophobic moiety.
The synthesis of the hydrophobic part, 2, from 3-(dimeth-
ylamino)-1,2-propanediol (1) is depicted in Scheme 1,
and it is identical to the first step of the synthesis of
DOTMA.15 Compound 3 was made by alkylation of 2
with N-(2-bromoethyl)phthalimide. The phthalimide
(Pht) group is readily removed by refluxing of 3 together
with hydrazine and methanol to give 4 with an 80% yield.
The free amine group of 4 is the site for linking of the
additional portion of the cationic head group.
Currently, cationic liposomes, which are formed from
either a cytofectin or a combination of a cytofectin and a
neutral lipid, show particular promise for the delivery of
therapeutic genes.10 They have many important qualities
such as being nonimmunogenic and nontoxic.
Cytofectins are positively charged amphiphiles, which are
made up of a cationic head group attached by a linker to a
hydrophobic moiety. The cytofectins are classified under
several subgroups.11,12 Among the subgroups, the DOT-
MA analogues are reported to give high transfection effi-
ciencies, and have achieved the most widespread use. The
organic synthesis of most DOTMA analogues has been
very well documented,13 which has certainly aided their
utility. The formulation of DNA with multivalent cationic
lipid, 2,3-dioleoyloxy-N-[2-(spermine-carboxamido)eth-
yl]-N,N-dimethyl-1-propanaminum chloride (DOSPA,
Figure 1) has proved to be a very promising transfection
system. Further investigations of structure–property rela-
tionships of DOSPA–DNA complexes and the mecha-
nism of their action are important, so sufficient quantities
of multivalent cationic lipid DOSPA are critical for future
research. Unfortunately, a synthesis of DOSPA has never
been reported in the literature.14 This prompted us to
OR
OH
RO
a
b
HO
N
N
68%
77%
2
1
RO
RO
O
N
N
RO
+
RO
+
c
N
O
NH2
80%
4
3
R =
7
8
Scheme 1 Reagents and conditions: a) xylene, NaH (2 equiv), oleyl
toluenesulfonate (2 equiv), 130 °C, 3 h; b) N-(2-bromoethyl)phthali-
mide (2 equiv), 110 °C, 18 h; c) hydrazine (2 equiv) flux together with
MeOH, 18 h.
SYNLETT 2006, No. 14, pp 2284–2286
0
1
.0
9
.2
0
0
6
Advanced online publication: 24.08.2006
DOI: 10.1055/s-2006-949636; Art ID: S02306ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
Synthesis of Cationic Lipid DOSPA
2285
The additional portion of the cationic head group, system study. The whole strategy highlights the utility of
tetra(tert-butoxycarbonyl)spermine-5-carboxylic acid the temporary amine-protecting group, phthalimide (Pht).
(Boc4SperCOOH), was synthesized in three steps as This temporary protecting group allows coupling of
described previously by Remy et al., 1994 (Scheme 2).16 various multivalent cationic headgroups with the lipid
Instead of using DMF as the solvent in the first step, part, which would facilitate the synthesis of new DOSPA-
methanol was used to perform the addition of acrylonitrile related cytofectins, and would allow detailed investiga-
to L-ornithine tetramethylammonium salt. The substitu- tion of their structure–activity relationships. Ultimately,
tion of methanol for DMF increased the yield to 85%. this may lead to the development of more efficient
After the hydrogenation and the protection of the amine cytofectins for gene delivery.
with a Boc group, the cationic head group precursor 8 has
only a free acid group, which can be coupled with the free
amine group of the hydrophobic part 4 under the standard
Acknowledgment
This work was supported by grant number NIH CA 96666 from the
National Institutes of Health, National Cancer Institute
coupling condition.
BocHN
H2N
CN
References and Notes
H2N
(1) Finkelstein, R.; Baughman, R. W.; Steele, F. R. Mol. Ther.
2001, 3, 3.
(2) Kayser, O.; Kiderlen, A. F. Pharm. Biotechnol. 2004, 249.
(3) Ozawa, K. Uirusu 2004, 54, 49.
HN
COOH
BocN
HN
COOH
a
b
c
COOH
COOH
85%
85%
76%
(4) McGarrity, G. J. Animal Cell Culture Techniques; Clynes,
M., Ed.; Springer: Berlin, 1998, 600–612.
(5) Amalfitano, A.; Parks, R. J. Curr. Gene Ther. 2002, 2, 111.
(6) Hauser, H.; Spitzer, D.; Verhoeyen, E.; Unsinger, J.; Wirth,
D. Cells Tissues Organs 2000, 167, 75.
HCl
NH2
NH
NBoc
NH
NC
(7) Suzuki, M.; Matsuse, T.; Isigatsubo, Y. Curr. Mol. Med.
2001, 1, 67.
NHBoc
NH2
(8) Friedmann, T. Sci. Am. 1997, 276, 96.
5
6
7
8
(9) Parker, A. L. J. Drug Targeting 2005, 13, 39.
(10) Miller, A. D. Medical and Biotechnology Applications, In
Microspheres, Microcapsules & Liposomes, Vol. 2; Citus
Books: London, 1999, 545.
Scheme 2 Reagents and conditions: a) MeOH, CH2CHCN, 24 h;
b) EtOH, Raney Ni, H2, 18 h; c) THF, Boc-ON.
(11) Bennett, M. J.; Aberle, A. M.; Balasubramanian, J. G.;
Malone, J. G.; Nantz, M. H.; Malone, R. W. J. Liposome
Res. 1996, 6, 545.
(12) Felgner, P. L.; Tsai, Y. J.; Felgner, J. H. Handbook of
Nonmedical Applications of Liposomes, Vol. 4; CRC Press:
Boca Raton, 1996, 43–56.
(13) Zabner, J. Adv. Drug Deliv. Rev. 1997, 27, 17.
(14) Miller, A. D. Angew. Chem. Int. Ed. 1998, 37, 1769.
(15) Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H.
W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen,
M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413.
As shown in Scheme 3, by using PyBOP, HOBt and DI-
PEA as standard coupling reagents, the hydrophobic part
4 and the protected cationic head group 8 can be linked to-
gether to give 9 with a yield of 70%. Treatment of 9 with
4 M HCl in dioxane led to the removal of the Boc group
of 9 to give the final product, DOSPA (10),17 with a yield
of 65%.
In conclusion, we have developed an efficient and
straightforward synthetic strategy to make sufficient mul-
tivalent cationic lipid DOSPA for nonviral gene delivery
+
H3N
BocHN
+
H2N
BocN
CONH
+
CONH
+
OR
OR
b
a
N
N
4 + 8
OR
OR
65%
70%
+
NH2
NBoc
R =
5 Cl–
R =
7
8
8
7
+ NH3
NHBoc
9
10
Scheme 3 Reagents and conditions: a) PyBOP, HOBt, DIPEA, CH2Cl2 12 h; b) 30% HCl in dioxane, 3 h.
Synlett 2006, No. 14, 2284–2286 © Thieme Stuttgart · New York

2286
Y. Li et al.
LETTER
(16) Remy, J. S.; Sirlin, C.; Vierling, P.; Behr, J. P. Bioconjugate
Chem. 1994, 5, 647.
Selected Data for Compound 10.
1H NMR (400 MHz, CD3OD): d = 5.38 (m, 4 H, CHCH),
3.92–3.02 (m, 30 H, CHCONH, NCH3, CH2O, CHO,
CH2N), 2.06–1.20 (m, 70 H, CH2, CH3, CH2CH2NH2,
CH2CH2NH, CH2CHCO). 13C NMR (100 MHz, CDCl3): d =
155.2 (CO), 130.1, 129.9 (CHCH), 80.6, 79.8, 78.9, 78.5
[C(CH3)3], 72.9 (CHO), 72.8, 71.2 (CH2NCH2), 68.7, 68.0
(OCH2), 60.9 (NCHCONH), 53.9, 52.8 (NCH3), 52.1
(CONHCH2CH2), 47.8–38.2 (CH2NCO), 32.1–22.1
(CH2CH2, CH2CH2NHCO, CH2CHCO), 14.3 (CH3).
MALDI-MS: m/z = 1072.1552 [M + H+].
(17) Selected Data for Compound 4.
1H NMR (400 MHz, CDCl3): d = 5.43 (m, 4 H CHCH),
4.11–3.89 (m, 7 H, OCH, OCH2), 3.59 (s, 6 H, 2 CH3), 3.51
(m, 6 H, NCH2CHO, NCH2), 2.00 (m, 8 H, CH2CHCH),
1.46–1.20 (m, 48 H, CH2), 0.93 (t, 6 H, J = 5.2 Hz, CH3). 13
C
NMR (100 MHz, CDCl3): d = 130.9, 129.7 (CHCH), 74.6
(CHO), 72.1, 69.5, 68.2 (CH2O), 65.8, 62.4 (CH2NCH2),
53.9, 53.5 (CH3), 43.1 (CH2NCO), 32.8-22.8 (CH2CH2),
14.3 (CH3). MALDI-MS: m/z = 664.6765 [M + H+].
Synlett 2006, No. 14, 2284–2286 © Thieme Stuttgart · New York