Synthesis of lipopolyhydroxylalkyleneamines for gene delivery

Article abstract of DOI:10.1016/j.bmcl.2006.01.120

Various bis(2-hydroxy-3-chloropropyl)alkylamines were synthesized by coupling primary amine with epichlorohydrin and utilized as a monomer to react with ethylenediamine (EDA), N,N′-dimethylethylenediamine (DMEDA), or tetramethylethylenediamine (TMEDA) to generate a series of lipopolyhydroxylalkyleneamines. The number- and weight-average molecular weight (M_n and M_w) and polydispersity index (M_w/M _n) of the lipopolyhydroxylalkyleneamines were dependent on reactant solvent and reaction temperature. The compounds with EDA as backbone have better transfection activity and lower toxicity than those with DMEDA and TMEDA as backbone.

Full text of DOI:10.1016/j.bmcl.2006.01.120

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2428–2432
Synthesis of lipopolyhydroxylalkyleneamines for gene delivery
Qun Li,a Guisheng Zhang,a Joie Marhefka,b Marina V. Kamenevab and Dexi Liua,*
aDepartment of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA
bMcGowan Institute for Regenerative Medicine and Department of Bioengineering, School of Engineering,
University of Pittsburgh, Pittsburgh, PA 15261, USA
Received 25 October 2005; revised 26 January 2006; accepted 26 January 2006
Available online 17 February 2006
Abstract—Various bis(2-hydroxy-3-chloropropyl)alkylamines were synthesized by coupling primary amine with epichlorohydrin
and utilized as a monomer to react with ethylenediamine (EDA), N,N0-dimethylethylenediamine (DMEDA), or tetramethylethylen-
ediamine (TMEDA) to generate a series of lipopolyhydroxylalkyleneamines. The number- and weight-average molecular weight (Mn
and Mw) and polydispersity index (Mw/Mn) of the lipopolyhydroxylalkyleneamines were dependent on reactant solvent and reaction
temperature. The compounds with EDA as backbone have better transfection activity and lower toxicity than those with DMEDA
and TMEDA as backbone.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Synthetic compounds such as cationic lipids1–3 and
polycations4–7 have been considered as potential carriers
for gene therapy due to their being easy to make and
highly flexible in delivering transgene of various sizes.
When mixing with plasmid DNA, these cationic com-
pounds form stable complexes in which DNA is tightly
condensed and protected from enzymatic degradation.
Previous studies have shown that various cationic lipids
and polycations are active in mediating gene transfer
into cells in culture and in animals.3 However, success
in applying these synthetic compounds to human gene
therapy has been limited primarily due to their poor
delivery efficiency compared to that of virus-based deliv-
ery systems. In addition, recent studies have demonstrat-
ed that polyethyleneimine (PEI), the most studied
polycation for gene delivery, is toxic to cells at higher
doses due to its high charge density and lack of biode-
gradability.8,9 Clearly, more efforts are needed to devel-
op new synthetic gene carriers and to improve activity of
existing compounds. Our ongoing research has focused
on the development of new synthetic compounds. In
the current study, we report the design, synthesis, and
gene transfer activity of lipopolyhydroxylalkyleneam-
ines, a novel class of compounds that bear the structural
features of cationic lipid and polycation.
Lipopolyhydroxylalkyleneamine
compounds
were
synthesized in two steps: preparation of various
bis(2-hydroxy-3-chloropropyl)alkylamine monomers10
and polymerization of monomers into lipopolyam-
ines.11 The procedures of Kim et al.12 were modified
to synthesize monomers 2a–2e (Scheme 1). Bis(2-hy-
droxy-3-chloropropyl)dodecylamine 2a, bis(2-hydroxy-
3-chloropropyl)dodecylaniline 2b, and bis(2-hydroxy-
3-chloropropyl)octadecylamine 2d were prepared by
reaction of epichlorohydrin 1 with dodecylamine, dod-
ecylaniline or octadecylamine, respectively. The same
strategy was used for synthesis of 2c and 2e using hex-
ylamine or methylamine to react with epichlorohydrin.
Monomers 2c and 2e were not stable at room temper-
ature and self-polymerization of 2e was detected by
1H NMR with a peak at 4.2–4.9 ppm. Thus, mono-
mers 2c and 2e were immediately used without further
purification. The yields of monomer synthesis are
summarized in Table 1.
Assembly of various bis(2-hydroxy-3-chloropropyl)
alkylamines into final product of lipopolyhydroxylalkyl-
eneamines (3a–3i) is outlined in Scheme 2. Reaction of
monomers of 2a, 2b, 2c, 2d or 2e with ethylenediamine
(EDA) afforded compounds 3a, 3b, 3c, 3d, and 3g.
Combination of 2e with N,N0-dimethylethylenediamine
(DMEDA) generated compound 3i. Compounds 3e,
3f, and 3h were produced by mixing N,N,N0,N0-tetra-
methylethylenediamine (TMEDA) with 2a, 2c or 2e,
respectively. The reaction of monomers of 2a, 2b, 2c,
or 2d with ethylenediamine in methanol/water at 40 ꢁC
Keyword: Gene delivery.
*
Corresponding author. Tel.: +1 412 648 8553; fax: +1 412 383
7436; e-mail: dliu@pitt.edu
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.120

Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2428–2432
2429
OH
R
N
OH
O
RNH2
Cl
Cl
Cl
s: ethanol
2a-e
1
2a: R = (CH2)11CH3
2c: R = (CH2)5CH3
2e: R = CH3
2b: R = p-C6H4-(CH2)11CH3
2d: R = (CH2)17CH3
Scheme 1. Synthesis of monomers 2a–2e.
Table 1. Synthetic yield of monomers
around 2000 at an initial monomer concentration [M]0
of 6 M. Polydispersity index (Mw/Mn) was 1.31–1.61
(Table 2), implying a well-controlled reaction. Structural
features of compounds 3a–3i are summarized in Table 2.
Monomer
R
Yielda (%)
2a
2b
2c
2d
2e
Dodecyl
4-Dodecylphenyl
Hexyl
86.5
91.5
92
The chemical structures of the synthesized compounds
were confirmed by 1H and 13C NMR and data are sum-
marized in Refs. 10 and 11. Figure 1 is a representative
1H NMR spectrum of lipopolyhydroxylalkyleneamine
using compound 3a as an example. It shows resonance
signal at 3.75–4.22 ppm due to the protons of CH–
OH. The single peak at 3.25 ppm corresponds to the
protons of hydroxyl group OH of polymers. A broad
multiple peak at 3.20–2.30 ppm corresponds to N–CH2
of the structural unit. The peak at 1.72–1.41 ppm is as-
signed to the N–CH2CH2 of dodecyl chain unit. Reso-
nance signals corresponding to the remaining protons
on methylene groups of the dodecyl chain appear at
1.29 ppm. The signal at 0.871 ppm is assigned to the
Octadecyl
Methyl
77
96
a Yield after column chromatography and based on the starting
primary amine.
for 3 days afforded a mixture that contains the desired
product, some oligomers (MW 600–750, 3–4 units),
unreacted monomers, and partially decomposed
compounds.
For compounds 3a–3f, copolymerization in n-butanol/
water at 90–95 ꢁC for 1–2 days resulted in compounds
with an average molecular weight of approximately
2000 as the major product, which was easily dissolved
in water with the exception of compound 3d. Metha-
nol/water mixture served as an ideal solvent for synthe-
sis of compounds 3g–3i. When butanol was used as
solvent for the reaction carried out with stirring at 85–
95 ꢁC for 1 h, a lot of solid was seen, which may be
either chloride salt of product with a low MW, or ethy-
lenediamine. If a mixture of methanol/water or butanol/
water (2:1 or 1:1) was used as reaction solvent, pale yel-
low and viscous slurry was always seen during polymer-
ization. The molecular weight of new compounds was
1
methyl group of the dodecyl chain unit. The H NMR
results indicate that the compounds with NH2 as an
end group are the overwhelming product.
Biological activities of the new compounds were evaluat-
ed using murine melanoma BL-6 cells according to an
established procedure.13 In brief, 5 · 105 cells/well in
48-well plates were seeded 24 h prior to transfection.
DNA/lipopolyamine complexes were prepared by mix-
ing various amounts of lipopolyhydroxylalkyleneamine
in serum-free medium with
a reporter plasmid
R1
R1
N
R2
N
R1
OH
R
N
OH
R
N
R1
N
OH R2
R2
N
R2
Cl
Cl
85-95 oC, 24-48 h
s: water/alcohol
OH
n
2a-e
3a-i
Scheme 2. Synthesis of lipopolyhydroxylalkyleneamines.
Table 2. Structural features of lipopolyhydroxylalkyleneamine
Compound
Monomer used
R
R1
R2
Mn
Mw
Mw/Mn
n
3a
3b
3c
3d
3e
3f
2a
2b
2c
2d
2a
2c
2e
2e
2e
Dodecyl
4-Dodecylphenyl
Hexyl
H
H
1831
1630
2072
1690
1943
1419
1500
1247
1396
2536
2168
2736
2265
2736
2404
2158
1783
1829
1.39
1.33
1.32
1.34
1.41
1.69
1.44
1.43
1.31
7
5
H
H
H
H
10
5
Octadecyl
Dodecyl
Hexyl
H
H
CH3
CH3
H
CH3
CH3
H
6
7
3g
3h
3i
Methyl
Methyl
Methyl
10
6
CH3
CH3
CH3
H
7

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Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2428–2432
Figure 1. 1H NMR spectrum in (D2O) of lipopolyhydroxylalkyleneamine 3a.
(pCMV-Luc) at the final DNA concentration of 4
lg/ml. The mixture was incubated at room temperature
for 15 min and then added to each well (1 lg of DNA/
well in 250 ll) after removal of the culture medium. Five
hour after transfection, 27.9 ll of fetal bovine serum
(FBS) was added to each well and incubation proceeded
for additional 19 h. Transfection medium was then re-
placed with fresh medium and cells were lysed 24 h later
with lysis buffer. Total proteins and luciferase activity in
cell lysates were determined. Figure 2 (left panel) shows
that transfection activity was much better for com-
pounds 3a, 3c, 3g, and 3h than for the rest of
compounds. Compounds with a secondary amine (com-
pound 3g), or quaternary amine (compound 3h),
exhibited similar transfection activity. Longer R groups
in compounds 3b, 3d–3f appeared less active compared
to that of compound 3g with R = CH3. The lowest activ-
ity was seen in compound 3f with R = (CH2)5–CH3 and
a quaternary amine. Compounds 3a and 3c showed sig-
nificant cytotoxicity, as reflected by decreased amount of
proteins recovered from each well of transfected cells
(Fig. 2, right panel). Overall, compound 3g (R = CH3,
R1 = R2 = H) has the structure exhibiting the best trans-
fection activity with relatively low toxicity.
In summary, a series of new lipopolyamines containing
hydroxyl, different amine groups (secondary, tertiary or
quaternary), and various alkyl groups were synthesized
Figure 2. Transfection activity (left) and cytotoxicity (right) of lipopolyhydroxylalkyleneamine 3a–3i. Cells (5 · 105 cells/well) were seeded in a 48-
well plate 24 h before the addition of DNA/compound complexes (1 lg DNA and various amounts of compounds). The level of luciferase gene
expression is expressed as mean of relative light units (RLU) per milligram of extracted protein. Cytotoxicity was indicated by the total amount of
proteins recovered from each well. Data represent means + SD (n = 3).

Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2428–2432
2431
2H), 1.57 (m, 2H), 1.39–1.11 (m, 18H), 0.82–0.92 (t, 3H);
13C NMR (CDCl3) d: 146.45, 145.76, 133.63, 132.72,
129.62, 129.54, 114.95, 113.16, 69.83, 69.12, 58.53, 56.43,
47.49, 47.05, 35.10, 35.04, 32.17, 32.03, 31.97, 29.93, 29.88,
29.80, 29.60, 22.94, 14.38.
Synthesis of 2c: epichlorohydrin 8.33 g (7.1 ml, 90 mmol)
was added dropwise to the solution of hexylamine 3 g (4 ml,
30 mmol) in ethanol (30 ml) at 5 ꢁC over 15 min. The
mixture was stirred at room temperature for 8 h and the
solvent was removed by vacuum desiccation. A transparent
and viscous liquid was obtained, which was further purified
by flash chromatography using hexanes/ethyl acetate (3:1,
Rf = 0.57) as the eluent to give the final product (12.5 g). 1H
NMR (CDCl3) d 0.845 (t, 3H), 1.215 (m, 6H), 1.416 (m, 2H),
2.493–2.72 (m, 6H), 2.95–3.41 (br s, 1H), 3.49–3.58 (m, 4H),
3.84–3.88 (m, 2H).
and characterized by NMR. Under our experimental
conditions, the number-average molecular weights
(Mn) of the compounds ranged from 1396 to 2072,
and the weight-average molecular weights (Mw) ranged
from 1783 to 2736. Biological tests in BL6 cells revealed
excellent but variable biological activities in gene deliv-
ery, depending on the backbone structure and the chain
length of the hydrophobic moiety. The detailed investi-
gations on structure–activity relation of these com-
pounds in vivo are in progress.
Acknowledgments
We thank Professor David Edwards for proofreading
of the manuscript. This work was supported in part by
the National Institutes of Health (R01-HL075542).
Synthesis of 2d: mixture of epichlorohydrin (10 g,
108 mmol) and octadecylamine (7 g, 25 mmol) in 30 ml
isopropyl ether and 60 ml ethanol was stirred at room
temperature overnight and concentrated under reduced
pressure. The crude product was isolated by chromatogra-
phy on a silica gel column with chloroform/methanol (14:1
v/v, Rf = 0.65) as the eluent to afford a white solid of 9.1 g.
1H NMR (CDCl3) d 3.91–3.86 (m, 2H), 3.61–3.50 (m, 4H),
3.49–3.18 (br s, 2H), 2.75–2.51 (m, 6H), 1.45 (br s, 2H), 1.24
(br s, 30H), 0.87 (t, 3H); 13C NMR (CDCl3) d: 69.30, 68.75,
58.89, 58.34, 56.13, 56.10, 47.64, 47.50, 32.15, 29.92, 29.88,
29.84, 29.76, 29.59, 27.51, 27.16, 27.09, 22.92, 14.34.
Synthesis of 2e: to 13 ml of ethanol epichlorohydrine
(13.1 g, 0.141 mmol) was added dropwise methylamine
(33% wt solution in absolute ethanol, 0.645 mmol) at ꢀ5 ꢁC.
The mixture was stirred at rt for 6 h and then at 4 ꢁC
overnight. The reaction mixture was then concentrated
under vacuum to afford the product as a colorless and
References and notes
1. Hirko, A.; Tang, F.; Hughes, J. A. Curr. Med. Chem.
2003, 10, 1185.
2. Ewert, K.; Slack, N. L.; Ahmad, A.; Evans, H. M.; Lin, A.
J.; Samuel, C. E.; Safinya, C. R. Curr. Med. Chem. 2004,
77, 133.
3. Liu, D.; Ren, T.; Gao, X. Curr. Med. Chem. 2003, 70,
1307.
4. Merdan, T.; Kopecek, J.; Kissel, T. Adv. Drug Deliv. Rev.
2002, 54, 715.
5. Kubasiak, L. A.; Tomalia, D. A. In Polymeric Gene
Delivery: Principles and Applications; Mansoor, M. A.,
Amiji, E., Eds.; CRC Press: Boca Raton, USA, 2005;
pp 133–157.
6. Haensler, J.; Szoka, F. C., Jr. Bioconjugate Chem. 1993, 4,
372.
7. Tang, M. X.; Redemann, C. T.; Szoka, F. C., Jr.
Bioconjugate Chem. 1996, 7, 703.
8. Li, Y.; Wang, J.; Lee, C. G.; Wang, C.; Gao, S. J.; Tang,
G. P.; Ma, Y. X.; Yu, H.; Mao, H. Q.; Leong, K. W.;
Wang, S. Gene Ther. 2004, 77, 109.
9. Kim, Y. H.; Park, J. H.; Lee, M.; Kim, Y. H.; Park, T. G.;
Kim, S. W. J. Controlled Release 2005, 103, 209.
10. Preparation of monomers. Synthesis of monomer 2a:
epichlorohydrin (3.9 ml, 50 mmol) was added to a solution
of dodecylamine (4.63 g, 25 mmol) in ethanol (13 ml) at
room temperature. The mixture was stirred for overnight
and the solvent was evaporated under vacuum. The crude
product was purified by flash chromatography on a silica
gel column with chloroform/methanol (14:1) as the eluent
to yield 2a as a white solid (8 g). 1H NMR (CDCl3) d:
0.861 (t, 3 H), 1.26 (single, 18H), 1.37–1.52 (brand, 2H),
2.74–2.51 (m, 6H), 3.25–3.48 (br s, 2H), 3.50–3.60 (m, 4H),
3.85–3.90 (m, 2H); 13C NMR (CDCl3) d: 69.30, 68.76,
58.87, 58.31, 56.12, 56.10, 47.64, 47.51, 32.13, 29.87, 29.85,
29.83, 29.76, 29.56, 27.51, 27.19, 27.13, 22.91, 14.34.
Synthesis of monomer 2b: mixture of epichlorohydrin
(1.16 g, 12.5 mmol), dodecylaniline (1.31 g, 5 mmol), and
ethanol (l ml) was stirred at room temperature for 18 h. A
white solid after removal of solvent under vacuum was
obtained and purified by flash chromatography using a
chloroform/isopropyl ether (10:0.7) eluent (Rf = 0.38),
resulting in 2.03 g white solid. The NMR results indicate
that this compound exists in two isomers. 1H NMR
(CDCl3) d: 7.08–7.05 (quat, 2H), 6.79–6.77 (d, 1H), 6.64
(d, 1H), 4.20–4.11 (m, 2H), 3.88 (d, 1H), 3.68–3.48 (m,
5H), 3.41–3.36 (m, 2H), 3.18–3.08 (m, 1H), 2.53–2.49 (m,
1
viscous liquid (96%). H NMR (CD3CN) d 2.28 (d, 3H),
2.41–2.56 (m, 4H), 3.51–3.57 (m, 2H), 3.61–3.66 (2H), 3.82–
3.89 (m, 2H).
11. Preparation of lipopolyhydroxylalkyleneamines: com-
pounds 3a–3f were synthesized by condensation of bis(2-
hydroxy-3-chloropropyl)alkylamine and ethylenediamine
(EDA) in 1-butanol/water (2:1) at around 95 ꢁC for 24–48 h
(compounds 3a–3d); or bis(2-hydroxy-3-chloropropyl)-
alkylamine and tetramethylenediamine in butanol/water
(1:1) with reflux overnight (compounds 3e, 3f). The reaction
mixture was concentrated under reduced pressure. After
washing with chloroform, acetone, and cooled methanol
to remove unreacted monomers and low molecular
weight oligomers, a crystalline amorphous solid was
obtained.
Compound 3a: 1H NMR (CD3OD) d 0.80 (t, 3H), 1.15–1.40
(br s 18H), 1.40–1.70 (br s 2H), 2.45–3.16 (br s, 14H), 3.82–
4.17 (br s, 2H).
Compound 3b: (D2O + DMSO) 1H NMR (D2O) d 0.81 (br
s, 3H), 0.98–1.28 (br s, 18H), 1.30–1.45 (br s, 2H), 2.20–2.49
(m, 2H), 2.65–3.88 (br s, 12H), 4.20–4.11 (m, 2H), 6.71 (br s,
2H), 7.08–7.05 (br s, 2H).
Compound 3c: 1H NMR (D2O) d 0.87 (br s, 3H), 0.95–1.42
(br s, 8H), 2.1–3.15 (14H), 3.81–4.10 (br s, 2H).
Compound 3d: 1H NMR (D2O) d 0.82 (br s, 3H), 0.95–1.52
(br s, 36H), 2.1–3.15 (14H), 3.51–4.15 (br s, 2H).
Compound 3e: 1H NMR (D2O) d 0.75–0.90 (br s, 3H), 1.15–
1.35 (br s, 18H), 1.38–1.52 (br s, 2H), 2.25–2.50 (br s, 6H),
2.60–2.8 (br s, 2H), 2.95–3.52 (m, 16H), 4.2–4.4 (br s,
2H).
1
Compound 3f: H NMR (D2O) d 0.85 (t, 3H), 1.24 (br s,
6H), 1.35–1.5 (br s, 2H), 2.48–2.65 (m, 6H), 2.74–2.95
(m, 2H), 3.10–3.65 (m, 18H), 3.95–4.35 (m, 2H).
Addition of monomer 2e to EDA/TMEDA/DMEDA
was accomplished using a 1:1 mixture of water/methanol

2432
Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2428–2432
3.10–2.65 (m, 2H), 2.65–2.42 (m, 4H), 2.40–2.30 (m, 3H),
as solvent at 85–95 ꢁC (Table 2) for 48 h to give
compounds 3g, 3h, and 3i which are hygroscopic white
solid. The compounds formed were purified as described
above.
2.28 (s, 1H).
Compound 3i: 1H NMR (H2O) d 4.24–4.00 (m, 2H), 3.06–
2.62 (m, 15H), 2.60–2.40 (m, 8H), 2.29 (s, 3H).
12. Kim, T. S.; Kida, T.; Nakatsuji, Y.; Ikeda, I. Langmuir
1996, 12, 6304.
Compound 3g: 1H NMR (H2O) d 4.18–3.96 (m, 2H),
3.10–2.45 (m, 17H).
Compound 3h: 1H NMR (H2O) d 4.48–4.31 (m, 1H),
4.26–4.14 (m, 0.5H), 4.10–3.91 (m, 1H), 3.68–3.14 (m, 15H),
13. Ren, T.; Zhang, G.; Liu, F.; Liu, D. Bioorg. Med. Chem.
Lett. 2000, 10, 891.