Bisethylnorspermine lipopolyamine as potential delivery vector for combination drug/gene anticancer therapies

Article abstract of DOI:10.1007/s11095-010-0197-4

Purpose: To design novel synthetic gene delivery system in which the carrier molecule functions dually as a carrier and a cytotoxic agent targeting dysregulated polyamine metabolism in cancer. Methods: Bisethylnorspermine (BENSpm) lipopolyamine was synthe

Full text of DOI:10.1007/s11095-010-0197-4

Pharm Res (2010) 27:1927–1938
DOI 10.1007/s11095-010-0197-4
RESEARCH PAPER
Bisethylnorspermine Lipopolyamine as Potential Delivery
Vector for Combination Drug/Gene Anticancer Therapies
Yanmei Dong & Jing Li & Chao Wu & David Oupický
Received: 12 April 2010 /Accepted: 15 June 2010 /Published online: 25 June 2010
#
Springer Science+Business Media, LLC 2010
ABSTRACT
ABBREVIATIONS
Purpose To design novel synthetic gene delivery system in
which the carrier molecule functions dually as a carrier and a
cytotoxic agent targeting dysregulated polyamine metabolism in
cancer.
Methods Bisethylnorspermine (BENSpm) lipopolyamine was
synthesized and its toxicity evaluated by MTS assay in MCF-7
and MCF-10A cells. Transfection activity was determined using
luciferase plasmid DNA.
BENSpm bisethylnorspermine
DOTAP
N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethy-
lammonium methylsulfate
FBS
SSAT
fetal bovine serum
spermidine/spermine N1-acetyltransferase
Results Asymmetrical lipid analogue of polyamine anticancer
drug BENSpm was synthesized using nucleophilic ring opening
of azetidinium ion. The synthesized LipoBENSpm showed
cytotoxicity comparable to that of parent BENSpm in human
breast cancer cell line MCF-7 but mediated 3–4 orders
magnitude higher transfection activity. Importantly, cytostatic
activity of BENSpm, typically in a low μM range, falls within a
relevant and typical concentration range required for efficient
gene delivery.
INTRODUCTION
Natural polyamines spermidine, spermine, and their di-
amine precursor putrescine are essential factors for growth
of eukaryotic cells. Polyamines play crucial roles in
numerous cellular processes important for cell growth and
survival, including association with nucleic acids, mainte-
nance of chromatin conformation, regulation of specific
gene expression, ion-channel regulation, maintenance of
membrane stability, and free-radical scavenging (1). Given
their natural function in DNA association, it is no surprise
that both spermidine and spermine have been investigated
for construction of synthetic gene delivery vectors (2,3).
Indeed, several of widely used lipid transfection reagents,
such as DOGS and DOSPA, are lipopolyamines with
spermine as the DNA binding functionality. Numerous
polycations based on spermine and spermidine have been
also synthesized and shown to exhibit favorable transfection
properties (4–6).
Conclusions These findings make gene delivery vectors based
on BENSpm promising candidates for combination drug/gene
approaches to the treatment of cancer.
.
.
KEY WORDS drug combination drug delivery
gene delivery lipid transfection
.
.
:
:
:
Y. Dong J. Li C. Wu D. Oupický (*)
Department of Pharmaceutical Sciences, Wayne State University
Detroit, Michigan 48202, USA
e-mail: oupicky@wayne.edu
Involvement of naturally occurring polyamines in cell
proliferation and differentiation prompted studies of the
polyamine pathway as a target for chemotherapeutic
intervention. Studies show that polyamine metabolism is
dysregulated in many types of cancer, leading to polyamine
Y. Dong
Department of Chemistry, University of Montreal
Montreal, Canada

1928
Dong, Li, Wu and Oupický
levels significantly higher than in normal tissues (7,8). It was
also suggested that the polyamine pathway is a distal
downstream target for a number of validated oncogenes
and that inhibition of polyamine synthesis disrupts the
action of those genes (1,9,10). The recognition that poly-
amines are required for cell growth and that their
metabolic pathway is frequently altered in cancers resulted
in the development of enzyme inhibitors for each step of the
polyamine biosynthetic pathway (11). In order to overcome
limitations of enzyme inhibitors, such as compensatory
upregulation, an alternative strategy has been to exploit the
self-regulatory nature of polyamine metabolism by devel-
oping polyamine analogues (12). A number of compounds
have been developed that show anticancer activity, includ-
ing symmetrically and asymmetrically terminally alkylated
polyamine analogues that can suppress the polyamine
biosynthetic enzymes, greatly induce the polyamine cata-
bolic enzymes, deplete natural polyamine pools, and inhibit
cell growth (13–15).
Among the most successful of the developed polyamine
analogues is N1,N11-bisethylnorspermine (BENSpm, also
known as DENSpm and BE333) (Fig. 1). BENSpm has
shown promising antitumor activity against a wide range of
cancers, including melanomas, ovarian, breast, and pan-
creatic cancers (16–19). BENSpm depletes intracellular
polyamines, downregulates both ornithine decarboxylase
and S-adenosylmethionine decarboxylase and upregulates
polyamine catabolism by inducing spermidine/spermine
N1-acetyltransferase (SSAT). The increase in polyamine
catabolism is then responsible for a mechanism of apoptotic
cell death observed in cells treated with BENSpm. In
addition, BENSpm has been successfully combined with
standard chemotherapeutic agents to produce synergistic
anticancer effect (20,21).
MATERIALS AND METHODS
Materials
Reagents and solvents were purchased from commercial
suppliers and were used without further purification unless
otherwise stated. Anhydrous THF and CH2Cl2 were dried
and distilled according to standard procedures. Ten
percent Pd/C was purchased from Aldrich (catalog No.
520888). All reactions were monitored by thin-layer
chromatography (TLC), which was viewed by UV light
at 254 nm and then stained with phosphomolybdic acid
(PMA) or ninhydrin in ethanol. IR spectra were recorded
on a Jasco FT/IR 4200 infrared spectrometer with ATR
PRO450-S attachment. Selected characteristic peaks are
reported in cm−1. NMR spectra were recorded on a
Varian FT-NMR Unity-300, Mercury-400 or Varian-
500 MHz Spectrometer. Chemical shifts (δ) are expressed
in ppm and are internally referenced (0.00 ppm for TMS
1
for H NMR and 77.0 ppm for CDCl3 for 13C NMR).
Mass spectra were recorded on a Waters ZQ2000 single
quadrupole mass spectrometer using an electrospray
ionization source. gWiz™ High-Expression Luciferase
(gWIZLuc) plasmid was purchased from Aldevron.
Synthesis
Synthesis of 3
The mixture of ethylbenzylamine 2 (1.35 g, 10 mmol), ethyl
acrylate (1.0 g, 10 mmol) and copper acetate monohydrate
(0.1 g, 0.5 mmol) in H2O/CH3CN (1:1) 40 mL was stirred
overnight at room temperature. Ethyl acetate (30 mL) was
added, and the organic layer was separated. The aqueous
phase was extracted with ethyl acetate (20 mL×3). The
combined organic layers were dried over anhydrous
Na2SO4. Evaporation gave the crude product and purifica-
tion by silica gel column gave the product 3 as colorless oil
(2.0 g, 85%). Rf 0.60 (n-hexane/ethyl acetate = 3/1). FTIR
(neat) 2972, 2803, 1732, 1369, 1194, 1163, 1045, 1027, 731,
697 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.15–7.17
(m, 5H), 4.08 (q, J=7.2 Hz, 2H), 3.54 (s, 2H), 2.79 (t, J=
7.2 Hz, 2H ), 2.48 (q, J=7.2 Hz, 2H), 2.42 (t, J=7.2 Hz,
2H), 1.19 (t, J=7.0 Hz, 3H), 1.00 (t, J=6.8 Hz, 3H); 13C
NMR (100 MHz, CDCl3) 172.2, 139.3, 128.3, 127.8, 126.4,
59.8, 57.6, 48.4, 46.8, 32.4, 13.8, 11.5.
The long-term goal of this study is to develop a novel
approach to the design of synthetic gene delivery systems
in which the carrier molecule functions dually as a
carrier and a cytotoxic agent targeting dysregulated
polyamine metabolism in cancer. Polyamine analogues
like BENSpm represent an ideal choice of the DNA-
binding functionality for the preparation of such delivery
systems due to their cationic nature and structural
simplicity. This report describes the method of synthesis
of asymmetrical lipopolyamine based on BENSpm and
initial evaluation of its cytoxocity and transfection
properties.
Synthesis of 4
N
N
N
N
A solution of amino ester 3 (5.1 g, 21.7 mmol) in dry THF
(10 mL) was added dropwise to a suspension of LiAlH4
(1.0 g, 26.3 mmol) in dry THF (40 mL) under nitrogen at
0°C. After addition, the mixture was stirred for 3 h under
H
H
H
H
BENSpm
Fig. 1 Structure of BENSpm.

Bisethylnorspermine Lipopolyamine for Anticancer Therapies
1929
reflux (monitored by TLC). The reaction mixture was cooled
to 0°C, and saturated NH4Cl solution was added slowly.
The mixture was stirred for another 30 min at room
temperature and then evaporated under vacuum. The
remaining aqueous solution was extracted with CH2Cl2
(30 mL×4). The combined organic layers were washed with
brine, dried over anhydrous Na2SO4, and concentrated
under vacuum; the residue was purified by silica gel column
to give the product 4 as colorless oil (3.93 g, 95%). Rf 0.53
(methanol/dichloromethane = 5/2). FTIR (neat) 3323,
dry CH2Cl2 (10 mL) was refluxed under nitrogen for 20 h.
During the heating, white precipitate appeared. After
completion of the reaction, water (15 mL) was added to
partition the mixture. Organic layer was separated, and
aqueous layer was extracted with CH2Cl2 (10 mL×3).
Combined organic layers were washed with brine, dried
over anhydrous Na2SO4, and evaporated to give the crude
product, which was purified by silica gel column to give the
structure of 7 (0.63 g, 90%) at Rf 0.1 and 8 ( 0.05 g, 5%) at
Rf 0.14 (methanol) as colorless oil. Data for 7: FTIR (neat)
3309, 2969, 2932, 2804, 1695, 1364, 1249, 1170, 730,
2933, 2809, 1452, 1051, 730, 697 cm−1 1H NMR
;
1
(400 MHz, CDCl3) δ 7.32–7.20 (m, 5H), 5.41 (bs, 1H),
3.69 (t, J=5.6 Hz, 2H), 3.54 (s, 2H), 2.62 (t, J=5.6 Hz, 2H ),
2.50 (q, J=7.2 Hz, 2H), 1.69 (quart, J=5.6 Hz, 2H), 1.06 (t,
J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) 138.2, 128.7,
128.8, 126.8, 63.8, 58.0, 53.0, 46.7, 27.6, 11.2; ESI MS
(m/z) Calcd for C12H19NO [M+H]+: 194.3, found: 194.4;
[M+Na]+: 216.3, Found 216.3.
698 cm−1; H NMR (400 MHz, CDCl3) δ 7.32–7.22 (m,
5H), 5.20 (bs, 1H), 3.53 (s, 2H), 3.18 (bq, 2H), 2.62–2.56 (m,
4H), 2.50 (q, J=7.2 Hz, 2H), 2.46 (t, J=7.2 Hz, 2H), 1.63
(m, 4H), 1.44 (s, 9H), 1.04 (t, J=7.4 Hz, 3H); 13C NMR
(100 MHz, CDCl3) 156.0, 139.9, 128.8, 128.1, 126.7, 78.8,
58.1, 51.3, 48.4, 47.9, 47.2, 39.4, 29.9, 28.4, 27.1, 11.7; ESI
MS (m/z) Calcd for C20H35N3O2 [M+H]+ 350.5, found:
350.5; [M+K]+ 388.3, Found: 388.3. Data for 8: FTIR
(neat) 3353, 2966, 2932, 2800, 1712, 1364, 1248, 1167, 730,
Synthesis of 5 and 6
1
697 cm−1; H NMR (400 MHz, CDCl3) δ 7.32–7.20 (m,
To a solution of amino alcohol 4 (3.93 g, 20.4 mmol) and
Et3N (14 mL, 102 mmol) in CH2Cl2 (20 mL) was added
methanesulfonyl chloride (MsCl) (3.15 g, 40.8 mmol) under
nitrogen at −10°C. The reaction mixture was stirred for 1 h
at −10°C and diluted with 2% Et3N in ethyl acetate
(20 mL). The mixture was immediately loaded to a short
deactivated silica gel column (2% Et3N in ethyl acetate) and
then eluted with 2% Et3N in ethyl acetate. Using methanol
as solvent to collect the eluate, the product of 5 (Rf 0.48,
EA/Hexane = 3/1) was partly decomposed to azetidinium
salt of 6 during evaporation. The product mixture of 5 and
6 was dissolved in CH2Cl2 (10 mL) and kept at room
temperature for 24 h and almost all transformed to 6 as
confirmed by TLC and NMR. Removing the solvent under
vacuum and drying with diethyl ether (5 mL) gave white
solid. Removal of diethyl ether under vacuum yielded the
hygroscopic product 6 (4.95 g, 100%), which was kept in
vacuum desiccator. mp 118–120°C. FTIR (neat) 3443,
10H), 5.51 (bs, 1H), 3.55 (s, 4H), 3.13 (bq, 2H), 2.49 (q, J=
7.1 Hz, 4H), 2.44–2.35 (m, 10H), 1.61–1.54 (m, 6H), 1.43 (s,
9H), 1.02 (t, J=6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3)
156.0, 139.9, 128.8, 128.1, 126.7, 78.7, 58.1, 52.7, 52.1,
51.4, 47.3, 40.0, 28.4, 26.6, 24.5, 11.7; ESI MS (m/z) Calcd
for C32H52N4O2 [M+H]+ 525.4, found: 525.4; [M+K]+
563.4. Found: 563.3.
Synthesis of 9
The mixture of 7 (0.349 g, 1 mmol), mesitylenesulfonyl
chloride (0.24 g, 1.1 mmol) and sodium hydroxide solid
(0.1 g, 2.5 mmol) in CH2Cl2 (10 ml) was stirred for 24 h
under nitrogen at room temperature. Water (10 mL) and sat.
aq. Na2CO3 (5 mL) were added at 0°C. Organic layer was
separated, and aqueous layer was extracted with CH2Cl2
(10 mL×3). The combined organic layers were washed with
brine, dried over anhydrous Na2SO4, and concentrated to
give the crude product, which was purified by silica gel
column to afford 9 (0.53 g, 100%) as colorless oil. Rf 0.60
(EtOAc). FTIR (neat) 3381, 2971, 2935, 1711, 1365, 1313,
1
2991, 1737, 1456, 1206, 1177, 1037, 766, 709 cm−1; H
NMR (400 MHz, CDCl3) δ 7.60–7.58 (m, 2H), 7.47–7.44
(m, 3H), 4.76 (q, J=8.8 Hz, 2H), 4.72 (s, 2H), 4.18 (q, J=
8.8 Hz, 2H), 3.49 (q, J=7.2 Hz, 2H), 2.77 (s, 3H), 2.60–2.53
(m, 2H), 1.44 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3) 131.6, 130.4, 129.4, 128.0, 61.9, 60.6, 54.5, 39.6,
14.1, 8.2; ESI MS (m/z) Calcd for azetidinium C12H18N+:
176.3, found M+ 176.4. Calcd for methanesulfonate
CH3O3S−: 95.0, found M− 94.9.
1
1248, 1147, 730, 698 cm−1; H NMR (400 MHz, CDCl3) δ
7.29–7.21 (m, 5H), 6.91 (s, 2H), 4.60 (bs, 1H), 3.40 (s, 2H),
3.22 (t, J=6.8 Hz, 2H), 3.11 (t, J=8.2 Hz, 2H), 3.05 (m,
2H), 2.57 (s, 6H), 2.35 (q, J=7.1 Hz, 2H), 2.26–2.24 (singlet
and triplet, 5H), 1.65 (quart, J=6.8 Hz, 2H), 1.54 (quart,
J=7.6 Hz, 2H), 1.42 (s, 9H), 0.93 (t, J=6.8 Hz, 3H); 13C
NMR (100 MHz, CDCl3) 155.8, 142.2, 139.9, 139.6, 133.2,
131.8, 128.5, 128.0, 126.6, 78.8, 57.8, 50.3, 47.1, 43.8, 42.9,
37.3, 28.3, 27.5, 24.8, 22.7, 20.8, 11.4; ESI MS (m/z) Calcd
for C29H45N3O4S [M+H]+: 532.3, found: 532.4; [M+K]+
570.3. Found: 570.4.
Synthesis of 7 and 8
The mixture of azetidinium methanesulfonate 6 (0.542 g,
2 mmol) and mono-Boc-1,3-diamine (0.766 g, 4.4 mmol) in

1930
Dong, Li, Wu and Oupický
Synthesis of 10 and 11
C31H49N3O6S2 [M+H]+ 624.3, found: 624.5; [M+K]+
662.3, Found: 662.4.
After three vacuum/hydrogen cycles to replace air inside the
reaction tube with hydrogen, the mixture of 9 (0.53 g,
1 mmol) and 10% Pd/C (0.265 g, 50% w/w) in methanol
(10 mL) was shaken at room temperature (22°C) under 50
psi hydrogen pressure for 2 days. The reaction mixture was
filtered, and the filter cake was washed with methanol. The
filtrate was concentrated to give crude product, which was
purified by a short silica gel column to yield the product 10
(0.40 g, 90%) as colorless oil at Rf 0.22 and 11 (0.05 g, 10%)
at Rf 0.25 (methanol). Data for 10: FTIR (neat) 3384, 2971,
Synthesis of 13
To a solution of 12 (0.623 g, 1 mmol) in anhydrous
CH2Cl2 (5 mL), trifluoroacetic anhydride (TFA) (5 mL) was
added slowly. The mixture was stirred for 4 h at room
temperature. The solvents were removed under vacuum,
and the residue was neutralized by 2 N aq. NaOH,
extracted with CH2Cl2 (15 mL×3). The combined organic
layers were washed with sat. aq. Na2CO3, dried over
anhydrous Na2SO4 and evaporated to give the crude
product, which was purified by silica gel column to yield
13 (0.523 g, 100%) as colorless oil. Rf 0.65 (10% triethyl
amine in methanol). ). FTIR (neat) 2976, 2937, 1603, 1456,
1311, 1145, 732, 694 cm−1; 1H NMR (400 MHz, CDCl3) δ
6.92 (d, 4H), 3.18 (t, J=7.2 Hz, 2H), 3.11 (q, J=7.1 Hz,
2H), 3.04 (q, J=6.8 Hz, 4H), 2.58 (t, J=7.6 Hz, 2H), 2.56
(s, 6H), 2.55 (s, 6H), 2.28 (s, 6H), 1.99 (bs, 2H), 1.70–1.66
(m, 2H), 1.59–1.55 (m, 2H), 0.97 (t, J=7.4 Hz, 3H); 13C
NMR (100 MHz, CDCl3) 142.2, 142.1, 139.7, 132.9,
132.8, 131.7, 131.6, 42.8, 42.3, 39.8, 38.6, 30.3, 24.9, 22.5,
22.4, 20.6, 12.4; ESI MS (m/z) Calcd for C26H41N3O4S2
[M+H]+ 524.3, found: 524.4; [M+K]+ 562.3, Found:
562.4.
1
2936, 1705, 1364, 1313, 1249, 1146, 732 cm−1; H NMR
(400 MHz, CDCl3) 6.95 (s, 2H), 4.57 (bs, 1H), 3.27–3.20 (m,
4H), 3.05 (q, J=6.0 Hz, 2H), 2.60 (s, 6H), 2.52 (q, J=
7.2 Hz, 2H), 2.48 (t, J=6.4 Hz, 2H), 2.30 (s, 3H), 1.72–1.62
(m, 4H), 1.42 (s, 9H), 1.03 (t, J=7.4 Hz, 3H); 13C NMR
(100 MHz, CDCl3) 155.7, 142.2, 139.7, 132.9, 131.7, 78.6,
46.3, 43.7, 43.1, 42.5, 37.2, 28.1, 27.4, 27.2, 22.5, 20.6,
14.9; ESI MS (m/z) Calcd for C22H39N3O4S [M+H]+
442.3, found: 442.4; [M+K]+ 480.3. Found: 480.3. Data for
11: FTIR (neat) 3390, 2974, 2937, 1698, 1365, 1313, 1249,
1
1147, 730 cm−1; H NMR (400 MHz, CDCl3) 6.95 (s, 2H),
4.71 (bs, 1H), 3.27–3.20 (m, 4H), 3.04 (q, J=6.0 Hz, 2H),
2.60 (s, 6H), 2.52 (q, J=6.8 Hz, 2H), 2.48 (t, J=6.4 Hz, 2H),
2.30 (s, 3H), 1.72–1.62 (m, 4H), 1.42 (s, 9H), 1.03 (t, J=
7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) 155.7, 142.2,
139.7, 132.9, 131.7, 78.6, 46.3, 43.7, 43.1, 42.5, 37.2, 28.1,
27.4, 27.2, 22.5, 20.6, 14.9; ESI MS (m/z) Calcd for
C23H41N3O4S [M+H]+: 456.3, found: 456.4; [M+K]+:
494.3, Found: 494.3.
Synthesis of 14 and 15
The mixture of 13 (0.575 g, 1.1 mmol), azetidinium
methanesulfonate 6 (0.271 g, 1 mmol) and Et3N (0.21 g,
2 mmol) in dry CH2Cl2 (10 mL) was refluxed for 24 h
under nitrogen. After completion of the reaction, water
(15 mL) and CH2Cl2 (10 mL) were added to partition the
mixture. Organic layer was separated, and aqueous layer
was extracted with CH2Cl2 (10 mL×3). Combined organic
layers were washed with sat. aq. Na2CO3, dried over
anhydrous Na2SO4, and evaporated to give the crude
product, which was purified by silica gel column to give the
structure of 14 (0.62 g, 88%) at Rf 0.20 and 15 (0.04 g,
5%) at Rf 0.22 (methanol/chloroform 10:1) as colorless oil.
Data for 14: FTIR (neat) 2972, 2936, 2872, 2806, 1603,
Synthesis of 12
The mixture of 10 (0.441 g, 1 mmol), mesitylenesulfonyl
chloride (0.24 g, 1.1 mmol) and sodium hydroxide solid
(0.1 g, 2.5 mmol) in CH2Cl2 (10 mL) was stirred for 24 h
under nitrogen at room temperature. Water (10 mL) and
sat. aq. Na2CO3 (5 mL) were added at 0°C. Organic layer
was separated, and aqueous layer was extracted with
CH2Cl2 (10 mL×3). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, concen-
trated and purified by silica gel column to give the structure
of 12 as colorless oil (0.623 g, 100%). Rf 0.35 (n-hexane/
AcOEt = 2/1). FTIR (neat) 3393, 2975, 2937, 1710, 1313,
1247, 1145, 694 cm−1; 1H NMR (400 MHz, CDCl3) δ 6.95
(s, 2H), 6.93 (s, 2H), 4.47 (bs, 1H), 3.13 (t, J=7.8 Hz, 4H),
3.05 (t, J=7.0 Hz, 4H), 2.99 (q, J=6.0 Hz, 2H), 2.57 (s, 6H),
2.55 (s, 6H), 2.29 (s, 6H), 1.71–1.67 (m, 2H), 1.61–1.57 (m,
2H), 1.42 (s, 9H), 0.98 (t, J=7.4 Hz, 3H); 13C NMR
(100 MHz, CDCl3) 155.6, 142.3, 142.1, 139.8, 139.7, 132.9,
132.8, 131.8, 131.7, 78.7, 42.9, 42.7, 42.3, 39.8, 37.1, 28.1,
27.1, 25.0, 22.5, 22.4, 20.6, 12.4; ESI MS (m/z) Calcd for
1454, 1313, 1146, 730, 696 cm−1; H NMR (400 MHz,
1
CDCl3) δ 7.30–7.22 (m, 5H), 6.91 (s, 4H), 3.52 (s, 2H), 3.12
(m, 4H), 3.04 (m, 4H), 2.55 (s, 12H), 2.49–2.45 (m, 4H),
2.41–2.36 (m, 4H), 2.28 (s, 3H), 2.27 (s, 3H), 1.70 (m, 2H),
1.55 (m, 4H), 1.03 (t, J=7.6 Hz, 3H), 0.99 (t, J=7.2 Hz,
3H); 13C NMR (100 MHz, CDCl3) 142.3, 142.2, 139.9,
139.8, 133.0, 131.8, 128.7, 128.0, 126.6, 58.0, 51.1, 48.2, 47.1,
46.8, 43.5, 43.0, 42.5, 40.0, 27.6, 27.0, 25.2, 22.7, 22.6, 20.8,
20.7, 12.6, 11.6; ESI MS (m/z) Calcd for C38H58N4O4S2
[M+H]+: 699.4, found: 699.6, [M+2H]2+ 350.6. Data for
15: FTIR (neat) 2966, 2936, 2871, 2799, 1603, 1453, 1315,

Bisethylnorspermine Lipopolyamine for Anticancer Therapies
1931
1147,730, 696 cm−1
;
1H NMR (400 MHz, CDCl3) δ
over anhydrous Na2SO4, and evaporated to give the crude
product. Purification by silica gel column gave the product
17 as colorless oil (0.27 g, 100%). Rf 0.35 (ethyl acetate).
FTIR (neat) 2973, 2931, 2802, 1686, 1414, 1364, 1248,
1153, 731 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.32–7.22
(m, 5H), 3.54 (s, 2H), 3.16 (broad m, 12H), 2.50 (q, J=
7.1 Hz, 2H), 2.42 (t, J=7.0 Hz, 2H), 1.70 (m, 6H), 1.45 (ds,
18H), 1.43 (s, 9H), 1.09 (t, J=7.2 Hz, 3H), 1.02 (t, J=
6.8 Hz, 3H). Coalescence peaks of carbon spectra were
found at room temperature (22°C), and thus higher
temperature (45°C) NMR was run on 500 MHz spectrom-
eter. 13C NMR (125 MHz, CDCl3) 155.43, 155.38, 155.33,
140.01, 128.71, 128.08, 126.68, 79.29, 79.12, 79.08, 58.21,
50.84, 47.38, 45.66, 45.01, 44.52, 41.81, 28.47, 27.67,
26.31, 13.59, 11.68; ESI MS (m/z) Calcd for
C35H62N4O6 [M+H]+: 635.5, found: 635.6; [M+K]+
673.5, Found: 673.6.
7.31–7.21 (m, 10H), 6.91 (s, 2H), 6.88 (s, 2H), 3.52 (s, 4H),
3.10 (q, J=7.2 Hz, 2H), 3.02 (m, 6H), 2.55 (s, 6H), 2.54 (s,
6H), 2.47 (q, J=6.4 Hz, 4H), 2.36 (t, J=7.6 Hz, 4H),
2.29–2.25 (m and ds, 10H), 2.18 (t, J=7.2 Hz, 2H), 1.70
(m, 2H), 1.49 (m, 6H), 1.01 (t, J=7.2 Hz, 6H), 0.98 (t, J=
7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) 142.3, 140.1,
140.0, 139.9, 133.2, 131.9, 128.8, 128.1, 126.7, 58.0,
51.8, 51.4, 51.3, 47.2, 44.1, 43.4, 42.6, 40.1, 25.5, 25.0,
24.3, 22.8, 22.7, 20.9, 20.8, 12.7, 11.7; ESI MS (m/z)
Calcd for C50H75N5O4S2 [M+H]+ 874.5, found: 874.6,
[M+2H]2+ 438.0.
Synthesis of 16
Phenol (0.235 g, 2.5 mmol) was added to the solution of 14
(0.35 g, 0.5 mmol) in dry dichloromethane (10 mL),
followed by 30% HBr in glacial acetic acid (1.3 mL) under
nitrogen at 0°C. The reaction mixture was stirred for 1 h at
0°C and then warmed up to room temperature (25°C).
After stirring for 24 h, the color of the solution turned
brown. Water (5 mL) was added at 0°C. The organic phase
was separated and extracted with 5 mL of water. The
combined aqueous phases were washed with dichloro-
methane (15 mL×5) until no more aromatic compounds
were detected by TLC. The brown aqueous layer was
evaporated under vacuum to get a solid residue, which was
washed with methanol (30 mL) to give the light yellow solid.
And then the lighter yellow solid was dissolved in water
(5 mL) and stirred with activated carbon (2 g) at 50°C for
10 min. The carbon was filtered off, and the solution was
evaporated under vacuum at 60°C to give the product 16
as white tetra-hydrobromide salt (0.31 g, 94%). Mp >230°C.
FTIR (neat) 3445, 3017, 2970, 1738, 1365, 1228, 1217,
Synthesis of 18 and 19
After removing the air in the reaction container with
hydrogen, the mixture of 17 (0.27 g, 0.43 mmol) and 10%
Pd/C (54 mg, 20% w/w) in methanol (10 mL) was shaken at
room temperature (22°C) under 50 psi hydrogen pressure
for 12 h. The reaction mixture was filtered, and the filter
cake was washed with methanol. The filtrate was concen-
trated to give crude product, which was purified by a short
silica gel column to yield the product 18 (0.21 g, 90%) as
colorless oil at Rf 0.13 and 19 (0.02 g, 10%) at Rf 0.15
(10% triethylamine in methanol). Data for 18: FTIR (neat)
3495, 2973, 2931, 1684, 1477, 1415, 1364, 1249, 1154,
1
771 cm−1; H NMR (300 MHz, CDCl3, 45°C) 3.26–3.15
(m, 12H), 2.67–2.57 (m, 4H), 1.77–1.67 (m, 6H), 1.45 (s,
27H), 1.01 (t, J=7.1 Hz, 6H); 13C NMR (75 MHz, CDCl3,
45°C) 155.5, 155.4, 155.3, 79.3, 79.2, 79.1, 46.9, 45, 44.8,
44.5, 44.1, 41.8, 29.0, 28.5, 27.7, 15.2, 13.6; ESI MS (m/z)
Calcd for C28H56N4O6 [M+H]+ 545.4, found: 545.4;
[M+K]+ 583.4. Found 583.4. Data for 19: FTIR (neat)
2974, 2931, 2791, 1685, 1477, 1415, 1365, 1249, 1156,
1
1204 cm−1; H NMR (400 MHz, D2O) 7.52 (m, 5H), 4.39
(d, 2H), 3.28–3.08 (m, 16H), 2.14 (m, 6H), 1.34 (t, J=
7.2 Hz, 3H), 1.27 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz,
D2O, spectral line broadening 0.1 Hz) 131.23, 130.56,
129.76, 129.11, 57.10, 48.64, 48.21, 44.97, 44.91, 44.88,
44.86, 44.14, 43.41, 22.99, 22.94, 20.90, 10.83, 8.47; ESI
MS (m/z) Calcd for C20H38N4 [M+H]+ 335.5, found:
335.5, [M+2H]2+: 168.4.
1
731 cm−1; H NMR (300 MHz, CDCl3, 45°C) 3.23–3.15
(m, 12H), 2.39 (q, J=7.1 Hz, 2H), 2.32 (t, J=7.2 Hz, 2H),
2.19 (s, 3H), 1.77–1.66 (m, 6H), 1.45 (s, 27H), 1.09 (t, J=
7.1 Hz, 3H), 1.03 (t, J=7.4 Hz, 3H); 13C NMR (75 MHz,
CDCl3, 45°C) 155.5, 155.4, 155.3, 79.3.79.2, 79.1, 54.7,
51.4, 45.6, 45.0, 44.5, 41.8, 41.5, 28.5, 27.7, 26.4, 13.6,
12.1; ESI MS (m/z) Calcd for C29H58N4O6 [M+H]+
559.4, found: 559.4; [M+K]+ 597.4. Found 597.3.
Synthesis of 17
The mixture of 16 (0.28 g, 0.43 mmol), Boc2O (0.37 g,
1.72 mmol) and Et3N (0.173 g, 1.72 mmol) in dry methanol
(10 mL) was stirred for 24 h under N2. Water was added,
and the mixture was evaporated under vacuum. The
remaining phase was partitioned between CH2Cl2
(20 mL) and water. The organic layer was separated, and
aqueous layer was extracted with CH2Cl2 (10 mL×3).
Combined organic layers were washed with brine, dried
Synthesis of 21
A suspension solution of 20 (22) (622 mg, 1 mmol) and
HOBt (203 mg, 1.5 mmol) in anhydrous CHCl3 (50 mL)
was added to the solution of EDCI (233 mg, 1.5 mmol) in

1932
Dong, Li, Wu and Oupický
anhydrous CHCl3 (20 mL) at 0°C, followed the mixture of
Et3N (152 mg, 0.21 mL, 1.5 mmol) and 18 (545 mg,
1 mmol) in CHCl3 (10 mL). The resulting mixture was
stirred at room temperature for 20 h and turned to clear
solution. The reaction mixture was then partitioned with
water (15 mL) at 0°C. The organic layer was separated,
and water layer was extracted with CHCl3 (10 mL×3).
Combined organic layers were washed with brine, dried
over anhydrous Na2SO4, and evaporated under vacuum to
give the crude product. The residue was dry-loaded to silica
gel column, and separation (Hexane/ethyl acetate 1:1) gave
the product 21 (1.0 g, 87%) as colorless oil. Rf 0.3
(Hexane/ethyl acetate 1:1). FTIR (neat) 2922, 2852,1737,
43.1, 42.7, 41.8, 31.9, 29.5 (m), 29.2 (m), 28.7, 28.1, 27.6
(d), 26.8, 26.7, 24.6, 23.1, 22.5, 13.2, 12.9, 10.4; ESI MS
(m/z) Calcd for C53H109N5O2[M+H]+ 848.85, found:
848.65, [M+2H]2+ 425.06.
Cytotoxicity Evaluation
Cytotoxicity of LipoBENSpm, control BENSpm and
DOTAP in human breast adenocarcinoma cell line
MCF-7 and in normal human breast epithelial cell line
MCF-10A was determined by MTS assay using a
commercially available kit (CellTiter 96® Aqueous Cell
Proliferation Assay, Promega). Four-thousand cells were
seeded in a 96-well plate, and after 24 the culturing
medium was removed and replaced with 200 μL of
RPMI/FBS with increasing concentration of a drug.
Medium was replaced every 48 h with fresh medium
containing the same concentration of the respective drug.
On day 6, the cell viabilities were determined by MTS
assay. The incubation medium was removed, and a
mixture of 100 μL of fresh medium and 20 μL of MTS
reagent solution was added to each well. The cells were
incubated for 1 h at 37°C in CO2 incubator. The
absorbance of each sample was then measured at
wavelength 505 nm to determine cell viability. The results
are expressed as mean percentage cell viability relative to
1
1691, 1643, 1466.1452, 1414, 1364, 1159, 772 cm−1; H
NMR (400 MHz, CDCl3) δ 3.38–3.16 (multiple broad
peak, 20H), 2.66–2.63 (bd, 4H), 1.85 (m, 2H), 1.72 (m, 4H),
1.55 (m, 2H), 1.48 (m, 2H), 1.43 (ds, 27H), 1.24 (bs, 66H),
1.08 (bt, 6H), 0.86 (t, J=6.8 Hz, 6H). 13C NMR (75 MHz,
CDCl3, 40°C) 171.7, 171.2, 155.3, 79.5, 79.2, 79.0, 47.9,
46.2, 45.6, 44.9 (multiple), 44.5, 43.8, 43.2, 41.8, 31.8,
29.6, 29.4, 29.3 (multiple), 29.2, 28.9, 28.5, 28.2, 27.7,
27.0, 22.6, 14.0, 13.6; ESI MS (m/z) Calcd for
C68H133N5O8 [M+H]+ 1149.01, Found 1148.88.
[M+K]+ 1186.97. Found 1186.82.
Synthesis of 1
untreated cells
SEM (n=4–8). IC50 values were
To the solution of 21 (0.322 g, 0.28 mmol) in anhydrous
CH2Cl2 (4 mL), TFA (2 mL) was added at 0°C. The
mixture was stirred for 2 h at room temperature. Most of
the solvents were evaporated under vacuum at 25°C. The
remaining solution (about 1 mL) was cooled to 0°C, and
2 mL of methanol was added to form white precipitate.
The white solid was obtained by centrifugation and washed
with small amount of methanol. The obtained white solid
was dried under vacuum to give the desired product as
trifluoroacetate salt. The white solid of LipoBENSpm
trifluoroacetate (0.16 g, 0.14 mmol) was suspended in
5 mL of CH2Cl2, and NaOH powder (0.1 g) was added at
0°C. The mixture was stirred for 2 h at room temperature,
and then anhydrous Na2SO4 was added. The filtration was
evaporated under vacuum, and methanol (2 mL), 2 N HCl
(0.21 mL, 0.42 mmol) was added at 0°C. The mixture was
stirred for 2 h at 0°C, and then freezing-dry and washing
with anhydrous ethyl ether gave the product LipoBENSpm
1 trihydrochloride salt as white solid. (0.13 g, 100%) Mp
180°C (dec) FTIR (neat) 2954, 2917, 2849, 2753, 1742,
obtained by Boltzmann Sigmoidal nonlinear regression.
Ethidium Bromide Exclusion Assay
The ability of LipoBENSpm and BENSpm to condense
plasmid DNA was confirmed by a standard ethidium
bromide (EtBr) exclusion assay by measuring changes in
EtBr/DNA fluorescence. Lipids were prepared in 10 mM
HEPES buffer (pH 7.4) at a concentration of 5 mg/mL.
Two mL of 20 μg/mL pDNA (gWiz-SEAP) solution was
mixed with EtBr (1 μg/mL). The fluorescence of EtBr/
DNA was measured using 540 nm excitation and 590 nm
emission and set to 100%. Relative fluorescence was
recorded following a stepwise addition of the lipids, and
the condensation curve for each lipid was constructed.
Agarose Gel Retardation Assay
DNA condensation ability of LipoBENSpm and BENSpm
was examined also by agarose gel electrophoresis. DNA
complexes were formed at different N/P ratios and
incubated for 30 min. Samples were loaded onto a 0.8%
agarose gel containing 0.5 μg/mL EtBr and run for 60 min
at 120 V in 0.5X TBE running buffer. The gel was
visualized under UV illumination on a Kodak Gel Logic
100 Imaging System.
1
1641, 1464, 1428, 1365, 1215 cm−1; H NMR (400 MHz,
CD3OD) 3.50 (m, 4H), 3.20–3.07 (m, 12H), 3.0 (t, J=
7.1 Hz, 2H), 2.71–2.70 (bd, 4H), 2.15 (m, 4H), 1.97 (m,
2H), 1.64 (m, 2H), 1.52 (m, 2H), 1.34 (t, J=7.2 Hz,
3H),1.29 (bs, 66H), 0.9 (t, J=7.4 Hz, 3H); 13C NMR
(100 MHz, CD3OD) 174.7, 172.6, 46.3, 45 (m), 44.5, 44.1,

Bisethylnorspermine Lipopolyamine for Anticancer Therapies
1933
Fig. 2 Structure of LipoBENSpm 1.
O
H
H
H
N
N
N
N
N
O
1 LipoBENSpm
Transfection activity
(Fig. 2). It consists of dioctadecylamine functionality
attached to a terminal amino group of BENSpm by amide
bond. The remaining three secondary amino groups are
free for the purpose of electrostatically binding with DNA.
The synthesis of LipoBENSpm 1 is dependent on the
availability of tri-protected BENSpm. Symmetrically and
asymmetrically substituted polyamine analogues have been
prepared by routes involving protection of internal amino
groups and asymmetrical introduction of terminal protect-
ing groups (13–18,23–28). The application of these
approaches to the synthesis of BENSpm analogues is
challenged by difficulties of selectively introducing protect-
ing groups to the terminal secondary amine in the presence
of three other secondary amines with similar reactivity.
Here, we proposed an effective synthesis of BENSpm with
three protected amines by nucleophilic ring opening of
azetidinium ion with amino group and employing appro-
priate amino protection and deprotection schemes.
The synthesis commenced with the Michael addition of
ethylbenzylamine 2 with ethyl acrylate using copper
acetate as catalyst in H2O/acetonitrile. The reaction
provided the intermediate amino ester 3 in good yield
(29) (Scheme 1). Reduction of 3 with lithium aluminium
hydride in tetrahydrofuran under reflux produced amino
alcohol 4 in high yield. Treatment of 4 with methane-
sulfonyl chloride in dichloromethane in the presence of
triethylamine at −10°C for 1 h gave the methanesulfonate
ester 5 as colorless oil in satisfying yield on TLC analysis.
However, decomposition of 5 was observed when the
reaction mixture was kept at room temperature, and a
All transfection experiments were conducted in 48-well plates.
Cells were seeded at a density of 40,000 cells/well 24 h before.
On the day of transfection, the cells were incubated with the
polyplexes prepared at different N/P ratios in 150 μL of
medium with or without 10% FBS. The dose of luciferase
pDNA was 0.4 μg per well. After 3 h of incubation, polyplexes
were completely removed, and the cells were cultured in
complete culture medium for 24 h prior to measuring
luciferase expression. The medium was then discarded, and
the cells were lysed in 100 μL of 0.5X cell lysis reagent buffer
(Promega). To measure the luciferase content, 100 μL of
0.5 mM luciferin solution was automatically injected into each
well of 20 μL of cell lysate, and the luminescence was
integrated over 10 s using BioTek Synergy 2 Microplate
Reader. Total cellular protein in the cell lysate was deter-
mined by the BCA (Bicinchoninic acid) protein assay using
calibration curve constructed with standard bovine serum
albumin solutions (Pierce). The transfection efficiency results
are expressed as Relative Light Units (RLU) per milligram of
cellular protein SD (n=4).
RESULTS AND DISCUSSION
Synthesis of LipoBENSpm
Asymmetrical lipopolyamine analogue of BENSpm, Lip-
oBENSpm 1, has been synthesized and characterized
Scheme 1 Synthesis of
intermediate 7.
ethyl acrylate
5 mol% Cu(OAc)2
H2O/CH3CN 1:1
O
LiAlH4, THF
reflux
Ph
NH
Ph
N
OH
Ph
N
O
2
3
4
CH3SO2Cl, Et3N
CH2Cl2, -10oC
O
CH2Cl2, rt
Ph
Ph
N
OMs
N
-O
S
O
5
6
Boc
H2N
N
H
CH2Cl2, reflux
Boc
Boc
Ph
N
H
N
N
H
Ph
N
2 N
N
H
7
8

1934
Dong, Li, Wu and Oupický
Scheme 2 Synthesis of
intermediate 10.
mesitylenesulfonyl chloride
NaOH solid, CH2Cl2, rt
Boc
N
Ph
N
N
7
H
Mts
9
H2, Pd/C
CH3OH
Boc
Boc
N
H
N
N
N
N
N
H
+
H
Mts
Mts
10
11
complete decomposition was observed within 24 h of
storage. Literature reports suggest that compounds con-
taining 3-aminopropyl methanesulfonate are unstable on
prolonged storage as free bases (30). Azetidinium meth-
anesulfonate is facile to form by intramolecular cyclization
reaction when treating hydroxypropyl amine with meth-
anesulfonyl chloride (31). Moreover, azetidinium four-
membered ring was also found in the salts of triflate
(32,33) and iodide (34). NMR spectroscopy of the
conversion compound shows the formation of four-
membered ring of azetidinium methanesulfonate 6, which
was also confirmed by ESIMS with an M+ signal at m/z
176.4 of cationic azetidinium moiety and M− at 94.9 of
methanesulfonate anion.
Azetidinium ion ring opening with nucleophiles such as
halides, alkoxides, phenoxides, amines and cyanide is
reported to be facile (35–37). Nucleophilic reaction of
azetidinium ring of 6 with mono-Boc-1,3-propanediamine
(38,39) in dichloromethane under reflux resulted in inter-
mediate 7 in 90% yield, with about 5% of 8 separated
chromatographically. Considering the high polarity of poly-
amines, the next step was to introduce a mesitylenesulfonyl
(Mts) protection group to the secondary amine 7 for
improved purification purposes (Scheme 2). The reaction
of 7 with mesitylenesulfonyl chloride (MtsCl) afforded 9 in
quantitative yield. Catalytic debenzylation of 9 with 10%
Pd/C (0.5:1, w/w) and hydrogen (50 psi) in methanol
provided 100% conversion with the isolation of 90% yield of
Scheme 3 Synthesis of tri-Boc
BENSpm 18.
mesitylenesulfonyl chloride
NaOH solid, CH2Cl2, rt
Boc
N
N
N
H
10
Mts
Mts
12
6
CH2Cl2, reflux
TFA, CH2Cl2
N
N
NH2
Mts
Mts
13
N
N
N
H
N
Ph
N
N
N
N
2
+
Mts
Mts
Mts
Mts
Ph
14
15
30% HBr in HOAc
phenol, CH2Cl2
.4HBr
Ph
N
H
N
H
N
H
N
14
16
H2, Pd/C
CH3OH
Boc2O, CH3OH
N
N
N
N
Ph
Boc
Boc
Boc
17
+
N
N
H
N
N
N
N
N
N
Boc
Boc
Boc
Boc
Boc
Boc
18
19

Bisethylnorspermine Lipopolyamine for Anticancer Therapies
1935
Scheme 4 Synthesis of
LipoBENSpm 1.
18
O
O
Boc
N
Boc
N
Boc
EDCI, HOBt
CHCl3, rt
OH
N
N
C17H35
C17H35
N
C17H35
C17H35
N
O
O
20
21
1) TFA-CH2Cl2 1:2
2) NaOH, CH2Cl2
3) 2N HCl, freeze-dry
O
H
H
H
N
N
N
N
C17H35
C17H35
N
O
.3HCl
1
10 and 10% yield of methylation product 11. We found that
more catalyst (10% Pd/C, 1:1 w/w) and longer reaction time
(4 days, 50 psi H2) increased the amount of 11 to 50%. It
was reported that N-methylation was observed in hydro-
genolysis of benzyl group in an alkaloid synthesis (40–42).
The proposed mechanism is that the catalyst of palladium
(40) or other precious metals such as ruthenium (43) and
iridium (44) can oxidize the adsorbed alcohol to the
corresponding aldehyde, which can be trapped by free
amine and then be reduced by hydrogen to N-alkylated
products. The hydrogenolysis was also run in ethanol and
trifluoroethanol solvents. At the same reaction conditions to
that in methanol, these two reactions were slower. Ten
percent yield of ethylation product was found in ethanol, and
a mixture including de-Boc products was found in
trifluoroethanol. No further efforts were extended to
improve this reaction. Methanol was used as hydro-
genolysis solvent in this work.
Next, another Mts group was introduced by treatment of
10 with MtsCl in dichloromethane to give the intermediate
12 quantitatively (Scheme 3). Subsequent removal of the
Boc group of 12 with trifluoroacetic acid gave the
corresponding disulfonamide 13 with free terminal amine.
Another nucleophilic ring opening reaction of azetidinium
ion of 6 with 13 in dichloromethane under reflux afforded
intermediate 14 in 88% yield and 5% yield of 15.
Fig. 4 Cytotoxicity of LipoBENSpm in (a) human breast cancer MCF-7
cells and (b) control normal MCF-10A breast cells. Metabolic activity was
measured by MTS assay following 144 h incubation of the cells with
increasing concentration of LipoBENSpm, BENSpm, and DOTAP (mean
S.D., n=3).
Fig. 3 1H-NMR and 13C-NMR of LipoBENSpm.3HCl in CD3OD.

1936
Dong, Li, Wu and Oupický
Deprotection of Mts groups of 14 with 30% HBr in acetic
acid and phenol in dichloromethane gave 16 as HBr salt in
94% yield. Subsequent protection of the three amino groups
using di-tert-butyl dicarbonate produced tri-Boc-protected
structure 17. 13C-NMR of 17 shows broad coalesced
resonance peaks when measured at 22°C. Performing the
NMR analysis at elevated temperature (45°C) improved
both the carbon and proton spectra resolution. Catalytic
debenzylation of 17 under H2 (10% Pd/C, 0.2:1, w/w)
yielded target structure tri-Boc BENSpm 18 in 92% yield,
with 8% of undesired methylation structure 19. Similar to
structure 17, coalescence of NMR spectra of 18 and 19
were found at room temperature, and well-resolved peaks
were obtained at 45°C.
N-succinyl-dioctadecylamine 20 was obtained by the
reaction of dioctadecylamine with succinic anhydride (22).
The combination of 20 and tri-Boc BENSpm 18 under
typical peptide-forming conditions (EDCI, HOBt) gave
the tri-Boc BENSpm lipid conjugate 21, which underwent
deprotection of Boc group (TFA, CH2Cl2) to yield the
desired structure 1 (Scheme 4). Considering its stability,
the final LipoBENSpm 1 was converted to hydrochloride
salt.
same time, another CH3 peak g of BENSpm mixed with
the CH2 peaks at about 1.3 ppm. The 13CNMR spectra of
LipoBENSpm shown in Fig. 4 indicate two amide carbon
atoms n and m (174.7 and 172.6 ppm). Three CH3 groups
in the structure are shown as a, g, h in the carbon spectra
(13.2, 12.8 and 10.4 ppm).
1.0E+10
B16F10
1.0E+09
-FBS
+FBS
1.0E+08
1.0E+07
1.0E+06
1.0E+05
1.0E+04
1.0E+09
1.0E+08
1.0E+07
1.0E+06
1.0E+05
1.0E+04
The structure of LipoBENSpm 1 was confirmed by
NMR in deuterated methanol (CD3OD) (Fig. 3). The
1HNMR peaks marked with letters a–k are assigned to the
corresponding hydrogen atoms of the lipid tail and cationic
head group. Due to the formation of the hydrochlorides,
the CH3 peak a of terminal amino group is shown as
expected down field at about 1.34 ppm, which overlapped
with the strong signal j of CH2 group of the lipid tail. At the
D2F2
-FBS
+FBS
LipoBENSpm
BENSpm
100
80
60
40
20
1.0E+08
1.0E+07
1.0E+06
1.0E+05
1.0E+04
1.0E+03
MCF-7
-FBS
+FBS
0
2
4
6
8
10
12
N/P ratio
Fig. 6 Transfection activity of LipoBENSpm in a panel of cancer cell
lines in the presence (+FBS) or absence (−FBS) 10% serum. Luciferase
activity was measured 24 h after 3 h incubation of cells with DNA
complexes (2.6 μg DNA/mL). Results are shown as mean RLU/mg of
cellular protein S.D. (n=3).
Fig. 5 DNA condensation by LipoBENSpm. (inset: gel retardation assay;
lane 1: free DNA; lanes 2–5: LipoBENSpm/DNA complexes prepared at
N/P 0.8, 1.6, 3.2, 6.4, respectively; lanes 6–9: BENSpm/DNA complexes
prepared at N/P 4, 8, 16, 32, respectively).

Bisethylnorspermine Lipopolyamine for Anticancer Therapies
1937
Gene Delivery Activity of LipoBENSpm
of LipoBENSpm. Transfection activity of LipoBENSpm
complexes was either higher or comparable to transfection
activity of complexes of both control lipids, DOTAP and
the structurally similar Transfectam.
The main goal of this study was to develop a method of
synthesis of asymmetrical BENSpm lipopolyamines and to
confirm their ability to effectively deliver genes. First, we
investigated cytotoxicity of LipoBENSpm in human breast
cancer cell line MCF-7 and in normal human breast
epithelial cell line MCF-10A (Fig. 4). LipoBENSpm
exhibited slightly enhanced cytotoxicity compared to
BENSpm in MCF-7 cells (IC50 0.36 0.04 μM vs. 1.03
0.10 μM) (Fig. 4a) and a significantly reduced cytotoxicity
in the normal MCF-10A control cells (8.73 0.60 μM vs.
0.19 0.01 μM) (Fig. 4b). In comparison, control lipid
DOTAP showed significantly lower cytotoxicity than Lip-
oBENSpm and BENSpm in both cell lines tested (IC50
33.84 1.84 μM in MCF-7 and 8.33 0.24 μM in MCF-
10A). The data in Fig. 4 do not allow reaching conclusion if
the observed effect of LipoBENSpm on cell viability and
the differences between LipoBENSpm and BENSpm in the
two cell lines are the result of a specific selective effect on
polyamine metabolism or demonstration of general toxicity
of cationic lipids. Available structure-activity studies suggest
that BENSpm may need to be released from the lipopoly-
amine in order to exert its intracellular effect. Detail
evaluation of the effect of BENSpm release on polyamine
catabolism is beyond the scope of this work and will be the
subject of our upcoming study.
CONCLUSIONS
In conclusion, we developed a method of synthesis of
asymmetrical analogues of polyamine anticancer drug
BENSpm. The synthesized LipoBENSpm shows cytotoxicity
comparable to that of parent BENSpm but mediates 3–4
orders of magnitude higher transfection activity. Importantly,
cytostatic activity of BENSpm falls within a relevant and
typical concentration range required for efficient gene
delivery. These findings make gene delivery vectors based
on BENSpm promising candidates for combination drug/
gene approaches to the treatment of cancer.
ACKNOWLEDGEMENTS
This work was supported by the National Institutes of Health
(R01 CA109711).
REFERENCES
The ability of LipoBENSpm to condense DNA was
investigated by ethidium bromide exclusion and agarose gel
retardation assays (Fig. 5). As expected, LipoBENSpm
efficiently condensed DNA above N/P ratio 2, while the
parent BESNpm could not retard DNA migration in the gel
or effectively exclude ethidium bromide even at N/P 16.
Transfection activity of the luciferase DNA complexes of
LipoBENSpm was measured in B16F10 mouse melanoma,
D2F2 mouse breast cancer, and MCF-7 human breast
cancer cell lines (Fig. 6). Transfection experiments were
conducted at three N/P ratios, and the results with
LipoBENSpm and BENSpm complexes were compared
with transfection activities of control DOTAP and Trans-
fectam lipoplexes. Even though total concentrations of
BENSpm and LipoBENSpm used in the transfection
experiments were above their IC50 values (Fig. 4), a
combination of the short incubation time (3 h vs. 144 h in
the cell viability experiment) and measuring the transfection
24 h after the incubation of complexes with cells ensured
that no significant toxicity was observed in the timeframe of
the transfection experiments. LipoBENSpm complexes
showed consistently high transfection activity in all three
cell lines both in the presence and absence of serum. As
expected based on the limited DNA condensation ability,
BENSpm complexes mediated low transfection activity,
typically 3–4 orders of magnitude lower than transfection
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