Synthesis and transfection efficiencies of new lipophilic polyamines

Article abstract of DOI:10.1021/jm0607101

A homologous series of lipophilic polyamines was synthesized and evaluated for DNA delivery and transfection efficiency. The series contained 1,4-butanediamine, 1,8-octanediamine, 2-[2-(2-amino-ethoxy)-ethoxy]-ethylamine, homospermidine, and homospermine

Full text of DOI:10.1021/jm0607101

308
J. Med. Chem. 2007, 50, 308-318
Synthesis and Transfection Efficiencies of New Lipophilic Polyamines
Richard Andrew Gardner, Mattias Belting,† Katrin Svensson,† and Otto Phanstiel IV*,‡
Department of Chemistry, P.O. Box 162366, UniVersity of Central Florida, Orlando, Florida 32816-2366, and Lund UniVersity, Department of
Oncology, Barngatan 2:1, S-221 85 Lund, Sweden
ReceiVed June 12, 2006
A homologous series of lipophilic polyamines was synthesized and evaluated for DNA delivery and
transfection efficiency. The series contained 1,4-butanediamine, 1,8-octanediamine, 2-[2-(2-amino-ethoxy)-
ethoxy]-ethylamine, homospermidine, and homospermine covalently attached via their N1 terminus to a
3,4-bis(oleyloxy)-benzyl motif. In addition, homospermidine and homospermine were also attached via amide
linkers. The homospermidine derivatives (i.e., benzyl tether 25 and benzamide tether 27) showed a 3-fold
and 4-fold respective enhancement in delivery of AlexaFluor-488-labeled DNA over the butanediamine
analogue 22. Homospermine derivative 26 was shown to inhibit 14C-spermine uptake (IC50 ∼10 µM), which
implied that 26 is able to compete effectively for polyamine recognition sites on the cell surface. This study
demonstrated that the number and position of the positive charges along the polyamine scaffold plays a key
role in DNA delivery and in determining the transfection efficiency.
Introduction
transfection than those with branched-, globular- or T-shaped
polar domains.21 Safinya et al.24 demonstrated with 324 that lipids
containing a higher number of positive charges had better
transfection efficiency within a series of liposomes containing
branched-polyamine headgroups. Other popular vectors 4-618b
are shown in Figure 2. Commercially available Lipofectamine
(LFAa) is a 3:1 mixture of 4 and 6.18c
Although a large number of cationic lipids have been
surveyed, a systematic study of how linear polyamine archi-
tectures effect transfection efficiency is still needed, especially
in light of the molecular recognition elements required to use
the polyamine transporter (PAT) for cellular entry.25-32 Our goal
was to perform the key crossover experiments needed to tie
these two fields together. Indeed, understanding how cationic
motifs are transported across the cell membrane is critical to
both enterprises.
Targeting a specific cellular transporter could provide cell-
selective transfection. For example, rapidly proliferating cancer
cells could be targeted via their PAT, which is often up-
regulated.28 Indeed, the ability to transfect a specific cell type
would have profound impact on a multitude of gene therapy
strategies.
Since many cancer cell lines have active polyamine transport-
ers, it is possible to target these cells using the molecular
recognition events involved in polyamine import.28 For example,
an anthracene-homospermidine conjugate was shown to be 10-
30 fold more toxic to B16 melanoma cells than to ‘normal’
melanocytes (Mel-A cells).28 A multitude of polyamine struc-
tures were previously screened for their high PAT selectivity
in Chinese hamster ovary cells (i.e., CHO and CHO-MG cells).28
Several linear polyamine architectures were identified, which
selectively targeted the PAT-active CHO cell line over its PAT-
inactive CHO-MG mutant.25-32 The discovery of homospermi-
dine, a 4,4-triamine, as a cell-selective ‘vector’ motif provided
the means to test the PAT-delivery system as a conduit for gene
delivery.26,28
With the information gained by the sequencing of the human
genome,1,2 gene therapy now holds promise for the treatment
of hereditary diseases and cancers.3,4 It is now possible to silence
a bad gene,5 turn on a needed gene,6 or install a new gene to
address particular cellular defects.7 However, a key requirement
for successful gene therapy is the efficient transfer of DNA to
specific cell types in ViVo. Viral vectors have proven to be very
efficient transfection agents and allow for the insertion of foreign
DNA into many cell types.8,9 However, the inherent problems
with viral vectors such as immunogenicity and the limited size
of the DNA plasmid that can be transferred has led to interest
in developing efficient nonviral vectors.10-14
Nonviral vectors are ideal because of their expected low
toxicity and immunogenicity, ability to transfer large strands
of DNA, and simpler synthetic preparation. Typically, these
vectors consist of a lipophilic component attached to a positively
charged, polar headgroup, through the use of a spacer or linking
motif, and are a mixture of neutral and positively charged lipids.
A wide range of cationic liposomes have been synthesized and
recently reviewed.15-17 The cationic headgroups typically found
in these liposome systems include quaternary ammonium salts,10
polylysine,18 polyguanadinium salts,19 polyarginine,20 and
polyamines.21
Polyamines were introduced into liposomes by Behr,22 when
it was realized that the naturally occurring polyamines such as
spermidine and spermine (Figure 1) could efficiently compact
DNA. Behr went on to synthesize one of the first transfection
vectors, 1 (DOGS11) and incorporated a branched polyamine
as the polar headgroup (Figure 2).11
Following this work, a number of research groups have looked
at the effect of changing the positively charged, polyamine
headgroup on cationic liposomes with respect to their efficiency
at transfecting DNA.15,16,21,23,24 Byk et al. showed that when
assayed in HeLa cells, compound 2,21 whose configuration of
the polar head is linear, was 5-10 times more effective at
a Abbreviations: PAT, polyamine transporter; BOC2O, di-tert-butyl
dicarbonate; LFA, Lipofectamine; DOSPA, 2,3-bis(oleyloxy)-N-2-(sper-
minecarboxamido)ethyl-N,N-diethyl-1-propanaminium trifluoroacetate; DOT-
MA, 1,2-bis(oleyloxy)propyl-3-trimethylammonium bromide; DOPE, di-
oleoylphosphatidylethanolamine.
* Corresponding author. Phone: (407) 823-5410, fax: (407) 823-2252.
e-mail: ophansti@mail.ucf.edu.
‡ University of Central Florida.
† Lund University.
10.1021/jm0607101 CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/22/2006

Transfection Efficiencies of Lipophilic Polyamines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 309
Figure 1. Native polyamines.
Figure 2. Structures of transfection lipids.
Scheme 1a
a Reagents: (a) K2CO3, KI, cyclohexanone; (b) KOH, EtOH; (c) oxalyl chloride.
In short, our aim was to combine these two areas of research
(PAT targeting and gene delivery) by attaching PAT-targeting
polyamine sequences28 to the aryl lipid motif (e.g., 324) in order
to facilitate DNA plasmid uptake. These materials were then
evaluated for their DNA-transporting ability as well as trans-
fection efficacy and compared to the commercially available
transfection reagent, Lipofectamine 2000.33
The 3,4-disubstituted benzene-containing lipids were cho-
sen due to the significant success of these lipids in earlier
transfection studies by Safinya and others.10,23,24 Indeed, this
structural element is present in the published transfection
agent 3 (Figure 2).10,23,24 The unsaturated unit within the
C18 chain was shown to prevent side-chain recrystallization
and conferred a high degree of flexibility upon the cationic
liposomes. These side chains were attached to the central
benzene core via ether linkages. An aldehyde or acid chlor-
ide in position 1 allowed for attachment of the polyamine
component. The polyamine scaffolds and controls were identi-
fied in earlier investigations of PAT-mediated drug deli-
very.25-29
Results and Discussion
Synthesis. A series of derivatives were synthesized with a
variety of linear polyamine headgroups. These cationic head-
groups contained from two to four positive charges. The
convergent synthetic route involved (a) the synthesis of the
hydrophobic lipid moiety, (b) the synthesis of the BOC-protected
amine head groups, and (c) coupling of the two separate
components followed by (d) the deprotection of the polyamine
moiety to provide the final compounds as HCl salts.
The first step was the synthesis of 3,4-bis(oleyloxy)benzal-
dehyde 8a by the O-alkylation of 3,4-dihydroxy benzaldehyde
7a with oleyl bromide (Scheme 1). Similarly, the bis-O-

310 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Gardner et al.
Scheme 2a
a Reagents: (a) BOC2O (0.33 equiv), MeOH, NEt3; (b) Br(CH2)3CN, K2CO3, CH3CN; (c) BOC2O (1.5 equiv), MeOH, NEt3; (d) H2, Raney Ni, EtOH,
NH4OH; (e) BOC2O, THF.
Scheme 3. Synthesis of the Lipid-Polyamine Conjugates 15-19a
a Reagents: (a) respective amine, CH2Cl2, MeOH; (b) NaBH4.
Scheme 4a
a Reagents: (a) 12 or 14, CH2Cl2, 1 M NaOH.
alkylation of ethyl 3,4-dihydroxybenzoate 7b with oleyl bromide
was used to form ethyl 3,4-bis(oleyloxy)benzoate 8b using the
method of Safinya et al.23,24 Subsequent cleavage of 8b with
KOH was carried out to give the acid 8c (Scheme 1) followed
by treatment with oxalyl chloride to give the desired acid
chloride 8d.
The final step to produce the desired lipid-polyamine
conjugates involved acidification of the amines with a solution
of anhydrous HCl in ethyl acetate.23,24 As shown in Scheme 5,
treatment of the penultimate compounds 15-21 with anhydrous
HCl/EtOAc provided the target HCl salts 22-28. By design,
the lipid portion of the conjugate (boxed structure in Scheme
5) was held constant throughout the series. This feature allowed
for later comparisons and an understanding of how the
polyamine component influenced DNA delivery.
As shown in Scheme 2, the next step in the synthesis in-
volved the generation of the BOC-protected polyamines: 9, 12,34
and 14. The mono BOC protection of diaminobutane gave
the amine 9 in a good yield. Sequential addition of bromo-
butyronitrile and BOC protection of the newly formed sec-
ondary amine, followed by reduction of the nitrile with Raney
Ni, gave masked triamine 12.34 Repetition of these three steps
on 12 gave tri-BOC-protected tetraamine 14 (via nitriles 13a
and 13b).
Biological Evaluation. Before conducting the transfection
studies, the series of conjugates (22-28) were first evaluated
for cytotoxicity in Chinese hamster ovary (CHO) cells. This
was an important step in determining what dose of lipid-
polyamine conjugate could be tolerated by the cell line. Ideally,
one would use a dose of the conjugate that is not cytotoxic to
the cell line to be transfected. This is an important caveat in
evaluating DNA delivery systems.
The coupling of the aldehyde 8a to a range of polyamines
was based on previous procedures for coupling of amines to
benzaldehyde derivatives.25-28 As shown in Scheme 3, the
reductive amination of 8a was achieved in two steps via in situ
generation of the imine (with a series of amines) followed by
reduction using NaBH4 to give the respective secondary amines
(15-19).
As shown in Scheme 4, the respective polyamines 1234 and
14 were coupled to acid chloride 8d to provide the BOC-
protected benzamide systems, 20 and 21.
As expected, there were significant differences in the aqueous
solubility of these new conjugates. DMSO was added in portions
to provide aqueous solutions of 22-28. Since DMSO itself is
toxic to CHO cells above 40 µM, stock solutions of each
conjugate were made in such a manner so that the total DMSO
concentration remained below 40 µM. This constraint limited
the amount of conjugate that could be dosed. Poorly soluble

Transfection Efficiencies of Lipophilic Polyamines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 311
Scheme 5a
Figure 3. DNA uptake activity by novel cationic lipids 22-28
(compared with Lipofectamine, LFA). CHO K1 cells were grown to
confluence in 24-well plates, rinsed with medium, and incubated for 4
h at 37 °C with 1 µg/mL Alexa Fluor-488-DNA complexed with 0.5
µg /mL (white bars), 2.5 µg /mL (gray bars), or 5 µg /mL (black bars)
of the respective cationic lipid. Cells were then extensively washed
with PBS and treated with trypsin, and cell pellets were again
extensively washed with PBS 1% BSA to completely remove nonspe-
cific fluorescence from the cell exterior. Finally, cells were analyzed
for Alexa Fluor-488-DNA uptake by flow cytometry on a FACSCalibur
(BD Biosciences) operated by Cell-Quest software. (*) During this
relatively short incubation (4 h), cells remained viable with 28. Note:
in converting from µg/mL to µM, the concentrations used were typically
approximately 0.6, 3.0, and 6 µM, respectively, and are tabulated
specifically in the Supporting Information. The negative control with
DNA only gave 3.8 ( 0.6 a.u. (data not shown).
a Reagents: (a) anhydrous HCl in ethyl acetate.
materials required higher DMSO levels, which in turn limited
the amount of material that could be dosed to cells in our toxicity
screen.
the 5 µg/mL dose. Nevertheless, DNA uptake experiments with
tetraamine 26 had over a 5-fold increase in DNA uptake (as
measured by fluorescence of the imported DNA probe) than
those conducted with diamine 22. Clearly, compound 26 was
more efficient in facilitating DNA delivery to cells.
Taking these factors into account, cytotoxicity screens were
performed to investigate the relative toxicity of each system.
Most materials were relatively nontoxic with 48 h IC50 values
g20 µM. Armed with this insight, cells were treated with e6.4
µM of the polyamine conjugate so as to avoid significant toxic
effects from the delivery agent itself. Indeed, at this dose, g90%
of cells survived transfection for all compounds except 28.
Indeed, compound 28 was very toxic (after 24 h incubation),
and even at the lowest dose used for transfection (0.5 µg/mL;
0.5 µM 28) killed 95% of the cells. As such, 28 was too toxic
for efficient transfection at the doses surveyed, and its data is
strongly biased by the few remaining cells, which survived.
It should be noted that the cytotoxicity screen was performed
with free lipopolyamine. The above data (g90% cell survival)
suggests that complex formation with DNA significantly at-
tenuates the cationic lipid cytotoxicity. This observation is
particularly relevant in the context of their use as transfection
agents.
Gene Transfer Studies. Armed with knowledge of the
cytotoxicity range of the series 22-28, CHO-K1 cells were
evaluated for DNA uptake using a fluorescently labeled DNA
(Alexa Fluor-488-DNA). As shown in Figure 3, cells were dosed
with the Alexa Fluor-488-DNA in the presence of increasing
concentrations (i.e., 0.5, 2.5, 5 µg/mL) of the respective
conjugate, 22-28. Each conjugate was as good (24, 25) or better
(26, 27) than the lipofectamine control (LFA, except 22 and
23) and facilitated uptake of the fluorescent DNA probe in a
concentration-dependent manner (µg/mL).
Interestingly, conjugate 27, which represents a butanediamine
motif similar to 22 except with the diamine placed further away
from the lipid tail, was a more efficient DNA delivery agent
than 22. In contrast, compound 23, which separated the
ammonium centers via an octanediamine had similar or lower
activity as 22 (depending on the dose). Insertion of the polyether
motif present in 24 maintained this eight-atom spacer, yet nearly
doubled the DNA delivery (DNA probe fluorescence) observed.
The PAT-selective homospermidine motif present in 25
resulted in a 3-fold increase in DNA delivery. However, this
outcome may simply be due to the presence of the additional
charge provided by the triamine motif present in 25. Indeed,
significant increases in DNA delivery were observed across the
homologous series 22, 25, and 26 which at 5 µg/mL gave 94,
281, and 504 fluorescence absorbance units (a.u.), respectively.
While the average amount of DNA taken up/cell shows wide
variations between the different compounds (Figure 3), all
compounds were able to deliver significant amounts of DNA
to virtually the entire cell population (Figure 4). However, in
terms of gene therapy, simple DNA delivery to the cell is
insufficient. There are other cellular barriers, which must be
traversed.
GFP Expression Studies. In order for the ‘therapy’ to be
effective, the DNA must escape from the endosome and enter
the cell’s nucleus, be transcribed to a regulatory, noncoding
RNA (RNAi) or to mRNA that is translated into its coded
protein. Therefore, we investigated the conjugate-assisted
expression of an eGFP DNA plasmid encoding for the green
fluorescent protein (GFP). CHO cells, which were successfully
transfected, were easily identified by their green fluorescence.
Control experiments conducted with only the eGFP DNA
plasmid (and no lipid carrier) gave virtually no fluorescence.
While the molecular weights in this series do range from 784
(22), 840 (23), 844 (24), 891 (25), 999 (26), 869 (27), and 976
g/mol (28), they are relatively close (within 10-22%) and allow
for general comparisons, especially in light of the large dif-
ferences observed in activity. For example, tetraamine 26, which
has the highest molecular weight of the series (999 g/mol), was
at a slightly lower concentration (5 µM) than 22 (6.4 µM) at

312 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Gardner et al.
Figure 6. The results obtained from the experiment described in Figure
5 were analyzed using a gated channel to determine the percentage of
cells that expressed significant levels of GFP as compared with cells
incubated with DNA plasmid only. A gate was set just above the
threshold measured with no DNA plasmid and no conjugate added (gate
value ) 5.5). All cells, which exhibited a fluorescence signal above
that level, were considered positive. Data are presented as the mean (
SD. Lipofectamine is denoted as LFA.
Figure 4. The results obtained from the experiment described by Figure
3 were analyzed using a gated channel to determine the percentage of
cells that had internalized significant amounts of DNA as compared
with cells incubated with DNA only. A gate was set just above the
threshold signal for control cells (DNA only with no lipid conjugate,
gate value ) 10.5). Note: Alexa Fluor-488-DNA complexed with 0.5
µg /mL (white bars), 2.5 µg /mL (gray bars), or 5 µg /mL (black bars)
of the respective cationic lipid was used, as indicated in Figure 3. Data
are presented as the mean ( SD. Lipofectamine is denoted as LFA.
black bars in Figure 4) gave 84% positive cells of the total cell
population remaining after the DNA uptake experiment; whereas
all the others (LFA, 22, and 24-28) gave typically >94%
positive cells. Using these data as benchmarks of DNA import
via each conjugate, the significantly lower % GFP positive cells
(23-67%) observed in Figure 6 suggests that intracellular
processing and nuclear delivery of DNA also depend on the
structure of the cationic lipid.
A closer analysis of the data revealed just how important these
latter two parameters (i.e., intracellular processing and nuclear
delivery) are for successful gene delivery. For example, although
26 was capable of delivering 3-fold more DNA to the cell than
the control LFA (black bars in Figure 3: 26: 504; LFA: 158
a.u.), the relative GFP expression/cell was only 2-fold higher
in Figure 5 (25 vs 13 a.u.) and the % of GFP positive cells was
only 1.5 fold higher in Figure 6 (67% vs 41%).
It cannot be excluded that maximum GFP expression occurs
at varying time points post-transfection for the different
compounds. Nevertheless, the data reflect an interesting cor-
relation between intracellular processing of internalized DNA
and the cationic lipid structure.
In summary, while a significant number of cells imported
the fluorescent DNA probe in the presence of the synthetic
conjugates (22-28), the amount of the DNA probe entering
each cell varied depending upon the conjugate used. The %
GFP positive cells of the total cell population roughly correlated
with the GFP expression/cell. Cells with low %GFP positive
cells (Figure 6: 23, 23%; 24, 25%) had low GFP expression/
cell (Figure 5: 23, 5; 24, 7). Cells with high %GFP positive
cells (Figure 6: 26, 67%) had high GFP expression/cell (Figure
5: 26, 25%). Indeed, the additional ‘intracellular barriers’
associated with successful GFP expression (as shown in Figure
6 in terms of % GFP positive cells) seemed to moderate the
large differences seen in the earlier DNA delivery study (Figure
3).
We speculated that 26 may be using the polyamine trans-
porter, PAT, for cellular entry. Indeed, as shown in Figure 7,
tetraamine 26 was shown to be a potent inhibitor of spermine
uptake (IC50 ∼ 10 µM) using a 14C-radiolabeled spermine
competition assay. This observation implied that 26 was able
to compete for the polyamine recognition sites on the cell surface
(e.g., PAT). Therefore, it is possible that certain lipophilic
polyamines, which present the correct polyamine ‘message’,
may be able to recognize and target cells expressing high levels
Figure 5. GFP expression results from transfection experiments. CHO
K1 cells were grown to approximately 50% subconfluence in 24-well
plates, rinsed with medium, and incubated for 4 h at 37 °C with 1
µg/mL eGFP DNA plasmid complexed with 0.5 µg /mL (white bars),
2.5 µg /mL (gray bars), or 5 µg /mL (black bars) of the respective
cationic lipid, as indicated above. Medium was then changed, and cells
were incubated for another 24 h to allow for GFP plasmid expression.
Cells were detached by trypsin treatment and analyzed for GFP
expression by FACS. Lipofectamine is denoted as LFA. The value
obtained with DNA plasmid only was subtracted from each entry above
(i.e., 1.2 ( 0.7 GFP expression with DNA only).
A gate or instrumental threshold was set based upon this low
background fluorescence. Fluorescence detected above this
background was considered a positive response.
The interpretation of the data in Figures 5 and 6 can be quite
subtle. For example, Figure 5 relates the amount of GFP
expression per cell and is a summary measurement of how well
the DNA ‘message’ was delivered and read and protein (GFP)
produced. While the magnitudes are different, the relative trends
are consistent with those observed in Figure 3. In this regard,
DNA delivery correlated with GFP expression.
Figure 6 revealed the % GFP positive cells observed in the
total cell population after the transfection experiment. This
information is a direct measure of transfection efficiency. Using
the 5 µg/mL dose for comparisons, 22 (38%), 25 (49%), and
27 (40%) were all comparable to the LFA control (41%) in terms
of transfection efficiency. Both 23 (23%) and 24 (25%) gave
lower values. The lone standout was tetraamine 26 (67%), which
had over 50% higher transfection efficiency than the LFA
control (41%).
Cells treated with conjugate 23 at the high dose (5 µg/mL,

Transfection Efficiencies of Lipophilic Polyamines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 313
plasmid DNA is critical for the future design of efficient
polyamine transfection agents.
Experimental Section
Materials and Methods. Silica gel (32-63 µm) and chemical
reagents were purchased from commercial sources and used without
further purification. All solvents were distilled prior to use. 1H and
13C NMR spectra were recorded at 300 and 75 MHz, respectively,
unless otherwise noted. TLC solvent systems are based on volume
%, and NH4OH refers to concentrated aqueous NH4OH. High-
resolution mass spectrometry was performed at the University of
Florida Mass Spectrometry facility.
ULYSIS AlexaFluor-488 (Molecular Probes) was used for
labeling of DNA as recommended by the manufacturer. All fine
chemicals were from Sigma. Lipofectamine 2000 was purchased
from Invitrogen. [14C]Spermine was purchased from Amersham
International, UK. Wild-type Chinese hamster ovary cells (CHO-
K1) were obtained from the ATCC (Manassas, VA). CHO cells
were routinely cultured in F12K nutrient mixture supplemented with
10% (v/v) fetal calf serum, 2 mM l-glutamine, 100 units/mL
penicillin, and 100 µg/mL streptomycin (growth medium) in a
humidified 5% CO2, 37 °C incubator.
DNA Uptake Studies. AlexaFluor-488-labeled DNA with or
without cationic lipid was mixed in serum-free F12K and added to
extensively rinsed cells grown in 24-well plates. Cells were then
incubated for 4 h at 37 °C. After removal of the incubation medium
and being rinsed with PBS, cells were detached with trypsin
followed by extensive washing with ice-cold PBS, 1% (w/v) bovine
serum albumin (BSA), to remove nonspecific fluorescence. Finally,
cells were suspended in PBS, 1% BSA, and analyzed for DNA
uptake by flow cytometry on a FACSCalibur (BD Biosciences)
operated by Cell-Quest software. Cells remained viable with all
compounds tested, including 28.
GFP Transfection Experiments. EGFP-encoding DNA plasmid
with or without cationic lipid was mixed in F12K media and
incubated with prerinsed, subconfluent cells in 24-well plates for
4 h, followed by another incubation period of 24 h in fresh growth
medium. Cells were then washed with PBS, detached by trypsin
treatment, dissolved in PBS, 1% BSA, and analyzed by FACS for
GFP expression. Note: severe toxicity was noted for compound
28 after the 24 h incubation period.
Figure 7. [14C]Spermine uptake inhibition by 26. Tetraamine 26 is an
efficient inhibitor of polyamine uptake. CHO-K1 cells were incubated
with 1 µM [14C]spermine (31 Ci/mol) for 20 min in serum free medium
supplemented with varying concentrations of tetraamine 26 as indicated
in the figure. After extensive rinsing, imported [14C]spermine was
determined by scintillation counting on cell lysates.
of polyamine transporters on their cell surface (e.g., cancer cells,
rapidly dividing tumors, etc.). However, as shown in this report,
delivery into the cell is just one step in successful gene
transfection. Nevertheless, our findings are an important first
step in developing ‘smart’ transfection agents.
Conclusions
This study demonstrated that the number and position of the
positive charges along the polyamine scaffold plays a key role
in DNA delivery and in determining the intracellular outcome
of the DNA import event, i.e., the transfection efficiency.
Although the Lipofectamine (LFA: 75% 4 and 25% 6 mixture)
has five positive charges (presumably a DNA delivery enhancing
feature) in one of its components (e.g., 4), it also has a neutral
component 6 present, which makes structural comparisons to
this control difficult beyond the dose-to-dose comparisons made
above. Future studies will look at the effect of neutral lipids
(like 6) in terms of transfection efficiency of polyamine-lipid
conjugates. Indeed, 6 has been shown to play a role in endosome
disruption and to facilitate transfection.35
Spermine Uptake Study in the Presence of 26. Subconfluent
CHO K1 cells (growing in 24-well plates) were rinsed three times
with F12K medium. The respective wells were dosed with the
corresponding dose of the spermine conjugate 26 and then incubated
for 20 min with [14C]spermine (31 Ci/mol). At this time point, the
cells were rinsed twice with F12K medium, three times with 1 mM
spermine (in F12K medium), and rinsed once with F12K medium.
The cells were lysed in 0.5 M NaOH for 1 h at 37 °C, and an
aliquot was removed and neutralized with 0.5 M HCl for scintil-
lation counting.37
Cell Cytotoxicity Studies. CHO cells were grown in RPMI
medium supplemented with 10% fetal calf serum, glutamine (2
mM), penicillin (100U/mL), and streptomycin (50 µg/mL). Cells
were grown at 37 °C under a humidified 5% CO2 atmosphere.
Aminoguanidine (2 mM) was added to the culture medium to
prevent oxidation of the drugs by the enzyme (bovine serum amine
oxidase) present in calf serum. Trypan blue staining was used to
determine cell viability before the initiation of a cytotoxicity
experiment. L1210 cells in early to mid log-phase were used. Cell
growth was assayed in sterile 96-well microtiter plates (Becton-
Dickinson, Oxnard, CA). CHO cells were plated at 2e3 cells/mL.
Drug solutions (10 µL per well) of appropriate concentration were
added after an overnight incubation for the CHO cells. After
exposure to the drug for 48 h, cell growth was determined by
measuring formazan formation from 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium using a Titertek Multiskan MCC/340
microplate reader for absorbance (540 nm) measurements.38 These
experiments allowed for a quick assessment of conjugate cytotox-
icity and facilitated determination of the proper dosing for the latter
In addition, this paper presented the first study that probed
the transfection phenomena with a PAT-selective homosper-
midine motif28 attached to a lipid cargo, e.g., 25. As expected,
compound 25 showed a 3-fold enhancement in DNA delivery
over its butanediamine analogue 22. Unfortunately, the DNA
delivery enhancement observed with 25 seemed to be muted
by the latter steps of the transfection process (e.g., intracellular
trafficking, nuclear delivery, etc.). As a result, 25 had only
slightly higher transfection efficiency than 22 (e.g., 25 and 22
gave 49% and 38% GFP positive cells in Figure 6, respectively).
This phenomenon was also observed with the two diamines,
27 and 22. Compound 27 showed greater than 4-fold enhance-
ment in DNA delivery over its butanediamine analogue 22.
Evidently, moving the diamine ‘message’ further away from
the lipid component enhanced the delivery characteristics of the
conjugate (Figure 3). Again, this potential delivery enhancement
by 27 was tempered by the latter steps of the transfection
process. Alas, nearly identical transfection efficiency was
observed for 27 and 22 (40% and 38% GFP positive cells in
Figure 6, respectively).
The ability of these polyamine transfection agents to both
target the polyamine transporter and deliver DNA was demon-
strated for tetraamine 26. However, cellular DNA delivery alone
was insufficient to predict successful transfection. Understanding
how different polyamine structures21,36 and neutral lipids like
635 enhance the intracellular trafficking and nuclear delivery of

314 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Gardner et al.
transfection and delivery experiments. Note: the cytotoxicity data
presented were performed with free lipopolyamine. Complex
formation with DNA significantly attenuated the cationic lipid
toxicity, which is relevant to their use as transfection agents.
3,4-Bis-octadec-9-enyloxy-benzaldehyde (8a). To a solution of
oleyl bromide (1.8 g, 5.4 mmol) in cyclohexanone (20 mL) was
added protocatachualdehyde (0.341 g, 2.5 mmol), potassium
carbonate (1.02 g, 7.4 mmol), and potassium iodide (0.05 g, 0.3
mmol). The suspension was stirred at 100 °C for 18 h under
nitrogen. Due to the light sensitivity of the oleyl bromide, the flask
was covered in aluminum foil. TLC (10% MeOH/CHCl3) showed
the reaction was complete. The hot reaction mixture was filtered
to remove some of the particulates, and the solvent was removed
in Vacuo. The residue was dissolved in CHCl3 (100 mL) and washed
with water (2 × 75 mL). The CHCl3 layer was dried over anhydrous
sodium sulfate and filtered and the solvent removed in Vacuo to
give a dark yellow oil. The oily residue was purified by flash
column chromatography (5% EtAc/hexane) to yield the product
8a as a white solid (1.08 g, 69%), Rf ) 0.3 (5% EtAc/hexane). 1H
NMR (CDCl3) δ 9.80 (s, 1H, CHO), 7.37 (m, 2H, phenyl), 6.93
(d, 1H, phenyl), 5.33 (t, 4H, olefinic), 4.06 (t, 2H, OCH2), 4.04 (t,
2H, OCH2), 2.01 (m, 8H, CH2CdC), 1.85 (m, 4H, CH2CH2O),
1.50-1.18 (m, 44H, 22 × CH2), 0.88 (t, 6H, CH3); 13C NMR
(CDCl3) δ 191.00, 155.34, 149.95, 130.14, 130.12, 129.97, 126.79,
111.89, 111.04, 69.36, 32.29, 30.16, 29.92, 29.90, 29.88, 29.72,
29.64, 29.62, 29.44, 29.36, 27.60, 26.37, 26.34, 23.09, 14.53; HRMS
(FAB): theory for C43H75O3 (M + 1), 639.5716; found (M + 1),
639.5755. Anal. (C43H74O3): C, H.
acetonitrile (25 mL) was added, and the resulting mixture stirred
at 50 °C for 24 h. TLC (1:10:89 NH4OH:MeOH:CHCl3) showed
that the reaction was 95% complete. The mixture was filtered to
remove most of the inorganic salts, and the acetonitrile was removed
in Vacuo to give a solid/oily residue that was purified by flash
column chromatography (1:5:94 NH4OH:MeOH:CHCl3) to yield
the product 10 as a clear oil (1.74 g, 61%), Rf ) 0.5 (1:10:89 NH4-
1
OH:MeOH:CHCl3); H NMR (CDCl3) δ 4.79 (br s, 1H, NHCO),
3.12 (q, 2H, CH2), 2.74 (t, 2H, CH2), 2.60 (t, 2H, CH2), 2.46 (t,
2H, CH2), 1.81 (quin, 2H, CH2), 1.57-1.37 (m, 13H, 2 × CH2, 3
× CH3).
(4-tert-Butoxycarbonylamino-butyl)-(3-cyano-propyl)-car-
bamic Acid tert-Butyl Ester (11).34 The amino-nitrile 10 (1.74 g,
6.8 mmol) was dissolved in a solution of triethylamine and methanol
(10% TEA in MeOH, 40 mL). A solution of di-tert-butyl dicar-
bonate (3.63 g, 0.017 mol) in methanol (20 mL) was added dropwise
to this mixture with vigorous stirring. The mixture was stirred at rt
overnight. TLC (1:10:89 NH4OH:MeOH:CHCl3) showed the tert-
butoxycarbonylation was complete. The methanol and TEA were
removed in Vacuo to yield an oily residue that was dissolved in
dichloromethane (100 mL) and washed with a solution of sodium
hydroxide (2.5 M, 3 × 30 mL) and water (2 × 30 mL). The organic
layer was dried over anhydrous Na2SO4, filtered, the solvent
removed in Vacuo and the oily residue purified by flash column
chromatography (40% EtAc/hexane) to give the product 11 as a
1
clear oil (2.10 g, 87%), Rf ) 0.45 (40% EtAc/hexane); H NMR
(CDCl3) δ 4.60 (br s, 1H, NHCO), 3.28 (t, 2H, CH2), 3.14 (m, 4H,
2 × CH2), 2.35 (t, 2H, CH2), 1.88 (quin, 2H, CH2), 1.58-1.28 (m,
22H, 2 × CH2, 6 × CH3).
3,4-Bis-octadec-9-enyloxy-benzoic Acid (8c). To a solution of
oleyl bromide (1.8 g, 5.4 mmol) in cyclohexanone (20 mL) was
added ethyl 3,4-dihydroxybenzoate 7b (0.45 g, 2.5 mmol), potas-
sium carbonate (1.02 g, 7.4 mmol), and potassium iodide (0.05 g,
0.3 mmol). The suspension was stirred at 100 °C for 18 h under
nitrogen. Due to the light sensitivity of the oleyl bromide, the flask
was covered in aluminum foil. The hot reaction mixture was filtered
to remove some of the particulates, and the solvent was removed
in Vacuo. The residue containing ester 8b was dissolved in a solution
of ethanol (20 mL) containing potassium hydroxide (0.8 g, 20
mmol) and refluxed for 4 h. The hot reaction mixture was added
to water (30 mL), and acidifying with 1 M HCl to pH 1 resulted in
the precipitation of a white solid. The solid 8c was filtered off and
washed several times with water. The crude acid was recrystallized
from ethanol (10 mL) to give the product 8c as a white solid (1.14
(4-Amino-butyl)-(4-tert-butoxycarbonylamino-butyl)-carbam-
ic Acid tert-Butyl Ester (12).34 The nitrile 11 (2.00 g, 5.6 mmol)
was dissolved in ethanol (100 mL). NH4OH (10 mL) and Raney
nickel (8 g) were added, and ammonia gas was bubbled through
the solution for 20 min at 0 °C. The suspension was hydrogenated
at 50 psi for 24 h. Air was bubbled through the solution, and the
Raney nickel was removed by filtering through a sintered glass
funnel, keeping the Raney nickel residue moist with solvent (EtOH)
at all times. The ethanol and NH4OH were removed in Vacuo, and
the oily residue was dissolved in CH2Cl2 and washed with 10%
aq, Na2CO3 (3 × 50 mL). The organic layer was dried over
anhydrous Na2SO4 and filtered and the solvent removed in Vacuo
to give the product 12 as a clear oil without further purification
(1.92 g, 95%), Rf ) 0.4 (1:10:89 NH4OH:MeOH:CHCl3); 1H NMR
(CDCl3) δ 4.65 (br s, 1H, NHCO), 3.14 (m, 6H, 3 × CH2), 2.69 (t,
2H, CH2), 2.35 (t, 2H, CH2), 1.58-1.26 (m, 26H, 4 × CH2, 6 ×
1
g, 70%); H NMR (500 MHz, CDCl3) δ 7.73 (dd, 1H, phenyl),
7.58 (d, 1H, phenyl), 6.89 (d, 1H, phenyl), 5.35 (t, 4H, olefinic),
4.05 (2 × t, 4H, 2 × OCH2), 2.01 (m, 8H, CH2CdC), 1.85 (m,
4H, CH2CH2O), 1.48 (quin, 4H, 2 × CH2), 1.41-1.19 (m, 40H,
20 × CH2), 0.88 (t, 6H, CH3).
1
CH3). The H NMR matched the literature spectrum of 12.34
(4-{tert-Butoxycarbonyl-[4-(3-cyano-propylamino)-butyl]-
amino}-butyl)-carbamic Acid tert-Butyl Ester (13a). To a solu-
tion the amine 1234 (1.32 g, 3.68 mmol) in anhydrous acetonitrile
(20 mL) was added potassium carbonate (1.7 g), and the suspension
was stirred at rt for 10 min. A solution of 4-bromobutyronitrile
(0.54 g, 3.68 mmol) in acetonitrile (10 mL) was added and the
resulting mixture stirred at 50 °C under nitrogen for 24 h. TLC
(1:10:89 NH4OH:MeOH:CHCl3) showed the reaction was 95%
complete. The mixture was filtered to remove most of the inorganic
salts, and the acetonitrile was removed in Vacuo to give a solid/
oily residue. The oil was redissolved in CH2Cl2 (70 mL) and washed
with saturated Na2CO3 (50 mL). The organic layer was separated
out, dried over Na2SO4, and filtered, the solvent was removed in
Vacuo, and the oily residue purified by flash column chromatog-
raphy using 7% MeOH/1% NH4OH/CH2Cl2 to give the product
Acid Chloride 8d. See compound 20 for experimental details.
(4-Amino-butyl)-carbamic Acid tert-Butyl Ester (9).34 1,4-
Diaminobutane (4.4 g, 0.05 mol) was dissolved in a solution of
triethylamine and methanol (10% TEA in MeOH, 110 mL). A
solution of di-tert-butyl dicarbonate (3.63 g, 0.017 mol) in methanol
(10 mL) was added dropwise to this mixture with vigorous stirring.
The mixture was stirred at RT overnight. The tert-butoxy-
carbonylation was complete as shown by TLC (4% NH4OH/
MeOH). The excess 1,4-diaminobutane, methanol, and TEA were
removed in Vacuo to yield an oily residue that was dissolved in
dichloromethane (100 mL) and washed with a solution of sodium
carbonate (10% aq, 2 × 100 mL). The organic layer was dried
over anhydrous sodium sulfate and filtered, the solvent removed
in Vacuo, and the oily residue purified by flash column chroma-
tography (1:10:89 NH4OH:MeOH:CHCl3) to give the product 9 as
a clear oil (2.23 g, 71%), Rf ) 0.38 (1:10:89 NH4OH:MeOH:
CHCl3). 1H NMR (CDCl3) δ 4.72 (br s, 1H, NHCO), 3.12 (q, 2H,
CH2), 2.70 (t, 2H, CH2), 1.57-1.30 (m, 13H, 2 × CH2, 3 × CH3).
[4-(3-Cyano-propylamino)-butyl]-carbamic Acid tert-Butyl
Ester (10).34 To a solution of the BOC protected diamine 9 (2.10
g, 0.01 mol) in anhydrous acetonitrile (50 mL) was added potassium
carbonate (5.14 g), and the suspension was stirred at RT for 10
min. A solution of 4-bromobutyronitrile (1.65 g, 0.01 mol) in
1
13a as a clear oil in 55% yield. H NMR (300 MHz, CDCl3) δ
4.60 (br s, 1H, NHCO), 3.15 (m, 6H, 3 × CH2), 2.74 (t, 2H, CH2),
2.62 (t, 2H, CH2), 2.46 (t, 2H, CH2), 1.83 (quin, 2H, CH2), 1.55-
1.39 (m, 26H, 4 × CH2, 6 × CH3); 13C NMR (CDCl3) δ 156.0,
155.6, 119.8, 79.3, 79.2, 53.6, 49.5, 48.1, 46.9, 40.4, 28.7, 28.6,
27.6, 27.3, 26.1, 25.9, 15.2.
(4-tert-Butoxycarbonylamino-butyl)-{4-[tert-butoxycarbonyl-
(3-cyano-propyl)-amino]-butyl}-carbamic Acid tert-Butyl Ester
(13b). A solution of di-tert-butyl dicarbonate (0.92 g, 0.42 mmol)
in methanol (10 mL) was added dropwise to the stirring solution

Transfection Efficiencies of Lipophilic Polyamines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 315
of 13a (0.12 g, 0.28 mmol) in 10% TEA/MeOH (10/80) at 0 °C
with vigorous stirring. The mixture was stirred at rt overnight. TLC
(1:10:89 NH4OH:MeOH:CHCl3) showed the tert-butoxycarbony-
lation was complete. The solvent was removed in Vacuo to yield
an oily residue that was dissolved in dichloromethane (100 mL)
and washed with a solution of saturated sodium carbonate (3 × 30
mL). The organic layer was dried over anhydrous sodium sulfate
and filtered, the solvent removed in Vacuo, and the oily residue
purified by flash column chromatography (CHCl3/MeOH 99:1) to
give the product 13b as a clear oil (95% yield). Rf ) 0.5 (CHCl3/
MeOH 99:2); 1H NMR (CDCl3) δ 4.63 (br s, 1H, NHCO), 3.28 (t,
2H, CH2), 3.14 (m, 8H, 4 × CH2), 2.64 (t, 2H, CH2), 1.88 (quin,
2H, CH2), 1.58-1.35 (m, 35H, 4 × CH2, 9 × CH3); 13C NMR: δ
155.9, 155.5, 79.9, 79.3, 79.0, 46.7, 45.8, 40.3, 28.6, 28.5, 27.5,
26.0, 25.7, 24.6, 14.9.
crude imine dissolved in CH2Cl2/MeOH (1:1, 10 mL). The solution
was cooled to 0 °C, and sodium borohydride (60 mg, 1.58 mmol)
was added in 15 mg portions over 30 min. TLC (10% EtAc/hexane)
showed the reaction to be complete after stirring overnight. The
solvent was removed in Vacuo and the crude solid dissolved in
CH2Cl2 (50 mL) and washed with sodium carbonate (10% aq, 3 ×
40 mL). The CH2Cl2 layer was dried over sodium sulfate and
filtered, the solvent was removed in Vacuo, and the solid residue
purified by flash column chromatography (1:10:89 NH4OH:MeOH:
CHCl3) to give the product 16 as a white solid (0.15 g, 63%), Rf
1
) 0.5 (1:10:89 NH4OH:MeOH:CHCl3). H NMR (CDCl3) δ 6.85
(s, 1H, aryl), 6.80 (m, 2H, aryl), 5.34 (t, 4H, olefinic), 3.97 (q, 4H,
OCH2), 3.69 (s, 2H, benzylic), 2.67 (t, 2H, CH2NH2), 2.60 (t, 2H,
CH2NH2), 2.01 (m, 8H, CH2CdC), 1.80 (m, 4H, CH2CH2O), 1.54-
1.18 (m, 56H, 28 × CH2), 0.88 (t, 6H, CH3); 13C NMR (CDCl3) δ
149.31, 148.20, 133.55, 130.11, 130.00, 120.59, 114.14, 114.07,
69.70, 69.50, 54.24, 49.83, 42.60, 34.20, 32.28, 30.46, 30.16, 29.92,
29.81, 29.71, 29.67, 27.73, 27.60, 27.22, 26.44, 23.08, 14.53; HRMS
(FAB): theory for C51H95N2O2 (M + 1), 767.7394; found (M +
1), 767.7377. Anal. (C51H94N2O2‚0.3H2O): C, H, N.
{4-[(4-Amino-butyl)-tert-butoxycarbonyl-amino]-butyl}-(4-
tert-butoxycarbonyl-amino-butyl)-carbamic Acid tert-Butyl Es-
ter (14). The nitrile 13b (1.33 g, 2.5 mmol) was dissolved in ethanol
(100 mL). Concentrated aq NH4OH (10 mL) and Raney nickel (6
g) were added, and ammonia gas was bubbled through the solution
for 20 min at 0 °C. The suspension was then hydrogenated at 50
psi for 24 h. Air was bubbled through the solution, and the Raney
nickel was removed by filtering through a sintered glass funnel
(keeping the Raney nickel residue moist at all times). The ethanol
and NH4OH were removed in Vacuo, and the oily residue dissolved
in CH2Cl2 and washed with 10 wt % aq sodium carbonate (3 × 50
mL). The organic layer was dried over anhydrous sodium sulfate
and filtered and the solvent removed in Vacuo. The oil was purified
by flash column chromatography (1:10:89 NH4OH:MeOH:CHCl3)
to give the product 14 as a clear oil (0.85 g, 64%), Rf ) 0.5 (1:
2-{2-[2-(3,4-Bis-octadec-9-enyloxy-benzylamino)-ethoxy]-eth-
oxy}-ethylamine (17). To a vigorously stirred solution of diami-
nodioxaoctane (0.23 g, 1.56 mmol, 5 equiv) in CH2Cl2/MeOH (3:
1, 5 mL) was added a solution of the aldehyde 8a (0.20 g, 0.33
mmol) in CH2Cl2/MeOH (3:1, 5 mL), dropwise over 1 h. The
resulting mixture was stirred at rt under an atmosphere of nitrogen
overnight. NMR showed the reaction to be complete when there
was no aldehyde peak present in the NMR spectrum. The solvent
was removed in Vacuo and the crude imine dissolved in CH2Cl2/
MeOH (1:1, 10 mL). The solution was cooled to 0 °C, and NaBH4
(60 mg, 1.58 mmol) was added in 15 mg portions over 30 min.
TLC (10% EtAc/hexane) showed the reaction to be complete after
stirring overnight. The solvent was removed in Vacuo and the crude
oil dissolved in CH2Cl2 (50 mL) and washed with sodium carbonate
(10% aq, 3 × 40 mL). The CH2Cl2 layer was dried over anhydrous
Na2SO4 and filtered, the solvent was removed in Vacuo, and the
oily residue purified by flash column chromatography (1:10:89 NH4-
OH:MeOH:CHCl3) to give the product 17 as a clear oil (0.168 g,
70%), Rf ) 0.45 (1:10:89 NH4OH:MeOH:CHCl3). 1H NMR
(CDCl3) δ 6.87 (s, 1H, aryl), 6.81 (m, 2H, aryl), 5.34 (t, 4H,
olefinic), 3.97 (q, 4H, OCH2), 3.72 (s, 2H, benzylic), 3.61 (m, 6H,
OCH2), 3.61 (t, 2H, OCH2), 2.86 (t, 2H, CH2NH2), 2.81 (t, 2H,
CH2NH2), 2.01 (m, 8H, CH2CdC), 1.80 (m, 4H, CH2CH2O), 1.54-
1.17 (m, 44H, 22 × CH2), 0.89 (t, 6H, CH3); 13C NMR (CDCl3) δ
149.31, 148.25, 133.11, 130.09, 129.99, 120.73, 114.26, 114.07,
73.57, 70.85, 70.57, 70.52, 69.69, 69.51, 54.02, 48.93, 42.01, 32.28,
30.15, 29.91, 29.80, 29.70, 29.67, 27.59, 26.45, 26.42, 23.07, 14.53;
HRMS (FAB): theory for C49H91N2O4 (M + 1), 771.6979; found
(M + 1), 771.6995; Anal. (C49H90N2O4): C, H, N.
(4-{[4-(3,4-Bis-octadec-9-enyloxy-benzylamino)-butyl]-tert-bu-
toxycarbonyl-amino}-butyl)-carbamic Acid tert-Butyl Ester (18).
To a vigorously stirred solution of the amine 12 (0.135 g, 0.38
mmol, 1.2 equiv) in CH2Cl2/MeOH (3:1, 5 mL) was added a
solution of the aldehyde 8a (0.20 g, 0.33 mmol) in CH2Cl2/MeOH
(3:1, 5 mL), dropwise over 20 min. The resulting mixture was stirred
at RT under an atmosphere of nitrogen overnight. NMR showed
the reaction to be complete when there was no aldehyde peak
present in the NMR spectrum. The solvent was removed in Vacuo
and the crude imine dissolved in CH2Cl2/MeOH (1:1, 10 mL). The
solution was cooled to 0 °C, and sodium borohydride (60 mg, 1.58
mmol) was added in 15 mg portions over 30 min. TLC (10% EtAc/
hexane) showed the reaction to be complete after stirring overnight.
The solvent was removed in Vacuo and the crude oil dissolved in
CH2Cl2 (50 mL) and washed with sodium carbonate (10% aq, 3 ×
40 mL). The CH2Cl2 layer was dried over sodium sulfate and
filtered, the solvent removed in Vacuo, and the oily residue purified
by flash column chromatography (3% MeOH/CHCl3) to give the
product 18 as a clear oil (0.24 g, 78%), Rf ) 0.3 (3% MeOH/
CHCl3). 1H NMR (CDCl3) δ 6.85 (s, 1H, aryl), 6.80 (m, 2H, aryl),
5.34 (t, 4H, olefinic), 4.60 (br s, 1H, NHCO), 3.97 (q, 4H, OCH2),
3.69 (s, 2H, benzylic), 3.13 (m, 6H, CH2) 2.63 (t, 2H, CH2NH),
1
10:89 NH4OH:MeOH:CHCl3); H NMR (CDCl3) δ 4.65 (s, 1H,
NHCO), 3.15 (m, 10H, 5 × CH2), 2.70 (t, 2H, CH2NH2), 1.58-
1.39 (m, 39H, 6 × CH2, 9 × CH3); 13C NMR (CDCl3): δ 155.9,
155.4, 79.2, 79.1, 47.0, 46.7, 40.3, 28.6, 28.5, 27.5, 26.1, 25.7;
HRMS (FAB): theory for C27H55N4O6 (M + 1), 531.4122; found
(M + 1), 531.4111; Anal. Calcd (C27H54N4O6‚0.1H2O): C, H, N.
[4-(3,4-Bis-octadec-9-enyloxy-benzylamino)-butyl]-carbam-
ic Acid tert-Butyl Ester (15). To a vigorously stirred solution of
the amine 9 (0.071 g, 0.38 mmol, 1.2 equiv) in CH2Cl2/MeOH (3:
1, 5 mL) was added a solution of the aldehyde 8a (0.20 g, 0.33
mmol) in CH2Cl2/MeOH (3:1, 5 mL), dropwise over 20 min. The
resulting mixture was stirred at rt under an atmosphere of nitrogen
overnight. 1H NMR showed the reaction to be complete when there
was no aldehyde peak present. The solvent was removed in Vacuo
and the crude imine dissolved in CH2Cl2/MeOH (1:1, 10 mL). The
solution was cooled to 0 °C, and NaBH4 (60 mg, 1.58 mmol) was
added in 15 mg portions over 30 min. TLC (10% EtAc/hexane)
showed the reaction to be complete after stirring overnight. The
solvent was removed in Vacuo and the crude oil dissolved in CH2-
Cl2 (50 mL) and washed with sodium carbonate (10% aq, 3 × 40
mL). The CH2Cl2 layer was dried over Na2CO3 and filtered, the
solvent removed in Vacuo, and the oily residue purified by flash
column chromatography (4% MeOH/CHCl3) to give the product
1
15 as a clear oil (0.18 g, 78%), Rf ) 0.2 (3% MeOH/CHCl3). H
NMR (500 MHz, CDCl3) δ 6.85 (s, 1H, aryl), 6.80 (m, 2H, aryl),
5.34 (t, 4H, olefinic), 4.78 (br s, 1H, NHCO), 3.97 (q, 4H, OCH2),
3.69 (s, 2H, benzylic), 3.13 (m, 2H, CH2) 2.63 (t, 2H, CH2NH),
2.01 (m, 8H, CH2CdC), 1.80 (m, 4H, CH2CH2O), 1.55 (m, 4H),
1.49-1.12 (m, 57H, 24 × CH2, 3 × CH3), 0.89 (t, 6H, CH3); 13C
NMR (CDCl3) δ 156.17, 149.34, 148.29, 133.15, 130.11, 130.00,
120.67, 114.18, 114.08, 79.23, 69.69, 69.52, 54.12, 49.22, 40.80,
32.28, 30.16, 29.92, 29.82, 29.71, 29.67, 28.81, 28.27, 27.68, 27.60,
26.44, 23.09, 14.54; Anal. (C52H94N2O4): C, H, N.
N1-(3,4-Bis-octadec-9-enyloxy-benzyl)-octane-1,8-diamine (16).
To a vigorously stirred solution of diaminooctane (0.23 g, 1.56
mmol, 5 equiv) in CH2Cl2/MeOH (3:1, 5 mL) was added a solution
of the aldehyde 8a (0.20 g, 0.33 mmol) in CH2Cl2/MeOH (3:1, 5
mL), dropwise over 1 h. The resulting mixture was stirred at RT
under an atmosphere of nitrogen overnight. NMR showed the
reaction to be complete when there was no aldehyde peak present
in the NMR spectrum. The solvent was removed in Vacuo and the

316 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Gardner et al.
2.01 (m, 8H, CH2CdC), 1.80 (m, 4H, CH2CH2O), 1.67-1.15 (m,
70H, 26 × CH2, 6 × CH3), 0.89 (t, 6H, CH3); 13C NMR (CDCl3)
δ 156.17, 155.71, 149.33, 148.25, 133.31, 130.11, 130.00, 120.62,
114.15, 114.08, 79.45, 69.70, 69.51, 54.13, 49.42, 47.34, 46.89,
40.58, 32.28, 30.14, 29.91, 29.80, 29.70, 29.67, 28.85, 28.79, 27.79,
27.70, 27.59, 26.42, 23.07, 14.53; HRMS (FAB): theory for
C61H112N3O6 (M + 1), 982.8551; found (M + 1), 982.8510; Anal.
(C61H111N3O6): C, H, N.
Anhydrous DMF (2 drops) and oxalyl chloride (0.3 mL) were added
in sequence, and the mixture was stirred for 1 h at 0 °C. The solution
was concentrated in Vacuo to give the crude acid chloride 8d. The
acid chloride 8d was dissolved in CH2Cl2 (10 mL) and added
dropwise to a solution of the amine 14 (0.195 g, 0.37 mmol, 1.2
equiv) dissolved in CH2Cl2 (10 mL) and 1 M NaOH (10 mL) that
had been cooled to 0 °C for 15 min. The reaction was stirred
overnight under N2 at room temperature. The water layer was
separated off. The CH2Cl2 layer was washed with Na2CO3 (10%
aq. 3 × 20 mL), dried over Na2SO4, filtered, and removed in Vacuo
and the oily residue purified by flash column chromatography (30%
EtAc/hexane) to give the product 21 as a clear oil (0.295 g, 83%),
(4-{[4-(3,4-Bis-octadec-9-enyloxy-benzylamino)-butyl]-tert-bu-
toxycarbonyl-amino}-butyl)-(4-tert-butoxycarbonylamino-butyl)-
carbamic Acid tert-Butyl Ester (19). To a vigorously stirred
solution of the amine 14 (0.20 g, 0.38 mmol, 1.2 equiv) in CH2-
Cl2/MeOH (3:1, 5 mL) was added a solution of the aldehyde 8a
(0.20 g, 0.33 mmol) in CH2Cl2/MeOH (3:1, 5 mL), dropwise over
20 min. The resulting mixture was stirred at RT under an
atmosphere of nitrogen overnight. NMR showed the reaction to be
complete when there was no aldehyde peak present in the spectrum.
The solvent was removed in Vacuo and the crude imine dissolved
in CH2Cl2/MeOH (1:1, 10 mL). The solution was cooled to 0 °C,
and sodium borohydride (60 mg, 1.58 mmol) was added in 15 mg
portions over 30 min. TLC (10% EtAc/hexane) showed the reaction
to be complete after stirring overnight. The solvent was removed
in Vacuo and the crude oil dissolved in CH2Cl2 (50 mL) and washed
with sodium carbonate (10% aq, 3 × 40 mL). The CH2Cl2 layer
was dried over sodium sulfate and filtered, the solvent removed in
Vacuo, and the oily residue purified by flash column chromatog-
raphy (3% MeOH/CHCl3) to give the product 19 as a clear oil
(0.319 g, 88%), Rf ) 0.25 (3% MeOH/CHCl3). 1H NMR (CDCl3)
δ 6.85 (s, 1H, aryl), 6.80 (m, 2H, aryl), 5.34 (t, 4H, olefinic), 4.60
(br s, 1H, NHCO), 3.97 (q, 4H, OCH2), 3.70 (s, 2H, benzylic),
3.14 (m, 10H, 5 × CH2) 2.64 (t, 2H, CH2NH), 2.01 (m, 8H, CH2Cd
C), 1.80 (m, 4H, CH2CH2O), 1.59-1.14 (m, 83H, 28 × CH2, 9 ×
CH3), 0.89 (t, 6H, CH3); 13C NMR (CDCl3) δ 156.12, 155.69,
149.33, 148.25, 133.28, 130.10, 129.99, 120.61, 114.14, 114.07,
79.40, 69.69, 69.50, 54.15, 49.47, 47.24, 46.98, 40.57, 32.27, 30.14,
29.90, 29.80, 29.69, 29.66, 28.85, 28.79, 27.77, 27.58, 26.44, 23.07,
14.53; HRMS (FAB): theory for C70H129N4O8 (M + 1), 1153.9810;
found (M + 1), 1153.9840; Anal. Calcd (C70H128N4O8): C, H, N.
(4-{[4-(3,4-Bis-octadec-9-enyloxy-benzoylamino)-butyl]-tert-
butoxycarbonyl-amino}-butyl)-carbamic Acid tert-Butyl Ester
(20). A solution of 3,4-bis-octadec-9-enyloxy-benzoic acid 8c (0.2
g, 0.31 mmol) in 2:1 dichloromethane /benzene (15 mL) was stirred
at 0 °C for 10 min. Anhydrous DMF (2 drops) and oxalyl chloride
(0.3 mL) were added in sequence, and the mixture was stirred for
1 h at 0 °C. The solution was concentrated in Vacuo to give the
crude acid chloride, 8d. Crude 8d was dissolved in CH2Cl2 (10
mL) and added dropwise to a solution of the amine 12 (0.132 g,
0.37 mmol, 1.2 equiv) dissolved in CH2Cl2 (10 mL) and 1 M NaOH
(10 mL) that had been cooled to 0 °C for 15 min. The reaction
was stirred overnight under N2 at room temperature. The water layer
was separated off. The CH2Cl2 layer was washed with Na2CO3 (10%
aq, 3 × 20 mL), dried over Na2SO4, filtered, and removed in Vacuo
and the oily residue purified by flash column chromatography (30%
EtAc/hexane) to give the product 20 as a clear oil (0.261 g, 86%),
1
Rf ) 0.20 (30% EtAc/hexane); H NMR (500 MHz, CDCl3) δ
7.42-7.20 (m, 2H, phenyl), 6.82 (br s, 0.5 H, NHCO), 6.80 (d,
1H, phenyl), 6.39 (br s, 0.5 H, NHCO), 5.35 (t, 4H, olefinic), 4.65
(m, 1H, NHCO), 3.99 (2 × t, 4H, 2 × OCH2), 3.42 (m, 2H, CH2),
3.13 (m, 10H, 5 × CH2), 2.00 (m, 8H, CH2CdC), 1.79 (m, 4H,
CH2CH2O), 1.53-1.17 (m, 83H, 28 × CH2, 9 × CH3), 0.88 (t,
6H, CH3); 13C NMR (CDCl3) δ 167.35, 156.16, 155.73, 151.88,
148.99, 130.13, 130.11, 127.37, 113.05, 112.41, 79.54, 69.54, 69.38,
47.03, 40.58, 39.97, 32.27, 30.14, 29.90, 29.77, 29.69, 29.66, 29.63,
29.60, 29.51, 28.85, 28.79, 27.78, 27.59, 26.40, 26.37, 23.07, 14.53;
HRMS (FAB): theory for C70H126N4O9 (M + Na), 1189.9407;
found (M + Na), 1189.9365; Anal. (C70H126N4O8): C, H, N.
N1-(3,4-Bis-octadec-9-enyloxy-benzyl)-butane-1,4-diamine, Di-
hydrochloride Salt (22). A concentrated solution of the amine 15
(0.158 g, 0.19 mmol) in ethyl acetate was added cooled to 0 °C. A
total of 4 mL of a freshly prepared saturated solution of HCl in
ethyl acetate was added dropwise and the solution stirred for 1 h
at room temperature during which time a white precipitate formed.
The ethyl acetate was removed in Vacuo, and the residue coevapo-
rated with ethyl acetate and chloroform to give the product 22 as
1
an off white powder (0.15 g, 98%); H NMR (500 MHz, CDCl3)
δ 9.50 (br s, 2H, R2N+H2), 8.22 (br s, 3H, RN+H3), 7.24 (s, 1H,
aryl), 7.05 (d, 1H, aryl), 6.82 (d, 1H, aryl), 5.34 (m, 4H, olefinic),
4.02 (q, 4H, OCH2), 3.93 (t, 2H, benzylic), 3.07 (m, 2H, CH2) 2.81
(m, 2H, CH2), 2.01 (m, 8H, CH2CdC), 1.95 (m, 2H, CH2), 1.85
(m, 2H, CH2), 1.78 (m, 4H, CH2CH2O), 1.45 (m, 4H, 2 × CH2),
1.38-1.20 (m, 40H, 20 × CH2), 0.89 (t, 6H, CH3); 13C NMR (300
MHz, CDCl3) δ 150.00, 149.37, 130.12, 130.09, 129.95, 122.58,
115.81, 113.49, 69.63, 69.38, 51.44, 51.06, 45.80, 39.63, 32.29,
30.24, 30.21, 30.16, 30.00, 29.93, 29.71, 27.61, 26.63, 26.49, 24.83,
23.75, 23.08, 14.54; HRMS (FAB): theory for C47H87N2O2 (M +
1), 711.6768; found (M + 1), 711.6827; Anal. (C47H88Cl2N2-
O4‚1.2H2O): C, H, N.
N1-(3,4-Bis-octadec-9-enyloxy-benzyl)-octane-1,8-diamine, Di-
hydrochloride Salt (23). A concentrated solution of the amine 16
(0.097 g, 0.13 mmol) in ethyl acetate was added cooled to 0 °C. A
total of 4 mL of a freshly prepared saturated solution of HCl in
ethyl acetate was added dropwise and the solution stirred for 1 h
at room temperature during which time a white precipitate formed.
The ethyl acetate was removed in Vacuo and the residue coevapo-
rated with ethyl acetate and chloroform to give the product 23 as
1
an off white powder (0.104 g, 98%); H NMR (CDCl3) δ 7.24 (s,
1
Rf ) 0.25 (30% EtAc/hexane); H NMR (500 MHz, CDCl3) δ
1H, aryl), 7.05 (d, 1H, aryl), 6.85 (d, 1H, aryl), 5.35 (m, 4H,
olefinic), 4.07 (q, 4H, OCH2), 3.95 (t, 2H, benzylic), 3.07 (m, 2H,
CH2) 2.79 (m, 2H, CH2), 2.05 (m, 8H, CH2CdC), 1.82 (m, 8H,
CH2CH2O, 2 × CH2), 1.58-1.21 (m, 52H, 26 × CH2), 0.89 (t,
6H, CH3); 13C NMR (300 MHz, CDCl3) δ 149.36, 148.78, 130.85,
130.33, 129.74, 122.23, 115.56, 113.45, 69.56, 69.32, 51.56, 51.15,
45.96, 39.86, 32.53, 30.44, 30.44, 30.23, 30.12, 30.00, 29.71, 27.61,
27.51, 27.10, 26.83, 26.69, 24.82, 23.77, 23.08, 14.54; HRMS
(FAB): theory for C51H95N2O2 (M + 1), 767.7394; found (M +
1), 767.7419; Anal. (C51H96Cl2N2O2‚0.3H2O): C, H, N.
7.50-7.20 (m, 2H, phenyl), 6.90 (br s, 0.5H, NHCO), 6.81 (d, 1H,
phenyl), 6.41 (br s, 0.5H, NHCO), 5.35 (t, 4H, olefinic), 4.68 (m,
1H, NHCO), 3.99 (2t, 4H, 2 × OCH2), 3.44 (m, 2H, CH2), 3.11
(m, 6H, 3 × CH2), 2.00 (m, 8H, CH2CdC), 1.79 (m, 4H, CH2-
CH2O), 1.53-1.17 (m, 70H, 26 × CH2, 6 × CH3), 0.88 (t, 6H,
CH3); 13C NMR (CDCl3) δ 167.32, 156.18, 151.84, 148.98, 130.11,
130.09, 127.32, 113.02, 112.39, 79.62, 79.32, 69.52, 69.36, 47.09,
40.49, 39.92, 32.26, 30.13, 29.89, 29.76, 29.68, 29.65, 29.62, 29.59,
29.50, 28.84, 28.77, 27.79, 27.58, 26.38, 26.36, 23.06, 14.51; HRMS
(FAB): theory for C61H109N3O7 (M + Na), 1018.8158; found (M
+Na), 1018.8123; Anal. (C61H109N3O7): C, H, N.
2-{2-[2-(3,4-Bis-octadec-9-enyloxy-benzylamino)-ethoxy]-eth-
oxy}-ethylamine, Dihydrochloride Salt (24). A concentrated
solution of the amine 17 (0.14 g, 0.18 mmol) in ethyl acetate was
added cooled to 0 °C. A total of 4 mL of a freshly prepared saturated
solution of HCl in ethyl acetate was added dropwise and the solution
stirred for 1 h at room temperature. The ethyl acetate was removed
in Vacuo and the residue coevaporated with ethyl acetate and
(4-{[4-(3,4-Bis-octadec-9-enyloxy-benzoylamino)-butyl]-tert-
butoxycarbonyl-amino}-butyl)-(4-tert-butoxycarbonylamino-bu-
tyl)-carbamic Acid tert-Butyl Ester (21). A solution of 3,4-Bis-
octadec-9-enyloxy-benzoic acid 8c (0.2 g, 0.31 mmol) in 2:1
dichloromethane/benzene (15 mL) was stirred at 0 °C for 10 min.

Transfection Efficiencies of Lipophilic Polyamines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 317
chloroform to give the product 24 as an off white powder (0.15 g,
98%); H NMR (CDCl3) δ 7.27 (s, 1H, aryl), 7.00 (d, 1H, aryl),
acetate was added cooled to 0 °C. A total of 5 mL of a freshly
prepared saturated solution of HCl in ethyl acetate was added
dropwise and the solution stirred for 1 h at room temperature during
which time a white precipitate formed. The ethyl acetate was
removed in Vacuo and the residue coevaporated with ethyl acetate
and chloroform to give the product 28 as a white powder (0.18 g,
1
6.82 (d, 1H, aryl), 5.33 (m, 4H, olefinic), 4.14 (m, 2H, benzylic),
4.01 (t, 2H, OCH2), 3.95 (t, 2H, OCH2), 3.87 (t, 2H, CH2), 3.78 (t,
2H, CH2), 3.70 (t, 4H, CH2), 3.25 (t, 2H, CH2) 2.93 (m, 2H, CH2),
2.02 (m, 8H, CH2CdC), 1.89 (m, 4H, CH2CH2O), 1.53-1.18 (m,
44H, 22 × CH2), 0.89 (t, 6H, CH3); 13C NMR (300 MHz, CDCl3)
δ 149.91, 149.62, 130.12, 130.00, 123.28, 122.61, 115.46, 113.63,
70.22, 70.09, 69.64, 69.40, 66.50, 65.98, 51.06, 45.08, 40.11, 32.28,
30.15, 29.92, 29.70, 27.60, 26.54, 26.43, 23.07, 14.54; HRMS
(FAB): theory for C49H91N2O4 (M + 1), 771.6973; found (M +
1), 771.6967; Anal. (C49H92Cl2N2O4‚0.4H2O): C, H, N.
N-(4-Amino-butyl)-N′-(3,4-bis-octadec-9-enyloxy-benzyl)-bu-
tane-1,4-diamine, Trihydrochloride Salt (25). A concentrated
solution of the amine 18 (0.17 g, 0.17 mmol) in ethyl acetate was
added cooled to 0 °C. A total of 4 mL of a freshly prepared saturated
solution of HCl in ethyl acetate was added dropwise and the solution
stirred for 1 h at room temperature during which time a white
precipitate formed. The ethyl acetate was removed in Vacuo and
the residue coevaporated with ethyl acetate and chloroform to give
the product 25 as a white powder (0.151 g, 98%); 1H NMR (CDCl3/
CH3OD 9:1) δ 7.13 (s, 1H, aryl), 7.02 (d, 1H, aryl), 6.85 (d, 1H,
aryl), 5.33 (m, 4H, olefinic), 4.05 (m, 2H, benzylic), 3.97 (t, 2H,
OCH2), 3.04 (m, 6H, 3 × CH2) 2.93 (m, 2H, CH2), 2.02 (m, 8H,
CH2CdC), 1.90 (m, 8H, 4 × CH2), 1.81 (m, 4H, CH2CH2O), 1.47
(m, 4H, 2 × CH2), 1.40-1.17 (m, 40H, 20 × CH2), 0.89 (t, 6H,
CH3); 13C NMR (300 MHz, D2O) δ 148.94, 148.33, 129.26, 129.19,
123.64, 122.52, 113.10, 68.50, 68.39, 49.96, 45.98, 45.43, 39.86,
31.28, 29.18, 29.11, 28.93, 28.86, 28.70, 28.62, 26.64, 26.59, 25.68,
25.62, 24.00, 22.54, 22.46, 22.11, 13.89; HRMS (FAB): theory
for C51H96N3O2 (M + 1), 782.7497; found (M + 1), 782.7495;
Anal. (C51H98Cl3N3O2‚0.6H2O): C, H, N.
1
98%); H NMR (CDCl3/CH3OD 9:1) δ 7.42 (m, 2H, aryl), 6.78
(m, 1H, aryl), 5.33 (m, 4H, olefinic), 4.03 (m, 4H, OCH2), 3.41
(m, 2H, CH2), 3.01 (m, 10H, 5 × CH2), 2.08-1.66 (m, 24H, 12 ×
CH2), 1.71 (m, 6H, 3 × CH2), 1.56-1.13 (m, 44H, 20 × CH2),
0.89 (t, 6H, CH3); HRMS (FAB): theory for C55H103N4O3 (M +
1), 867.8025; found (M + 1), 867.8022; Anal. Calcd for (C55H105
-
Cl3N4O3‚1.5H2O): C, H, N.
Acknowledgment. The authors wish to thank Dr. Jean-Guy
Delcros (Univ. of Rennes 1 in Rennes, France) for his help (with
OP) in determining the cytotoxicity profiles of the new
conjugates, and Dr. Navneet Kaur (UCF), who provided the
NMR characterization of compounds 13a, 13b, and 14. Partial
support was provided by the Broad Medical Research Program
of the Eli and Edythe L. Broad Foundation (OP), the Swedish
Cancer Fund (MB), the Swedish Research Council (MB), and
the Crafoordska Foundation (MB).
Supporting Information Available: Elemental analysis data
for compounds 8a and 14-28, conversions of doses into µM
concentrations for transfection experiments, sample fluorescent
images from transfection studies with eGFP plasmid, and 1H NMR
spectra for 8a, 8c, and 15-28. This material is available free of
charge via the Internet at http://pubs.acs.org.
N-[4-(4-Amino-butylamino)-butyl]-N′-(3,4-bis-octadec-9-eny-
loxy-benzyl)-butane-1,4-diamine, Tetrahydrochloride Salt (26).
A concentrated solution of the amine 19 (0.253 g, 0.22 mmol) in
ethyl acetate was added cooled to 0 °C. A total of 5 mL of a freshly
prepared saturated solution of HCl in ethyl acetate was added
dropwise and the solution stirred for 1 h at room temperature during
which time a white precipitate formed. The ethyl acetate was
removed in Vacuo and the residue coevaporated with ethyl acetate
and chloroform to give the product 26 as a white powder (0.215 g,
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98%); H NMR (CD3OD) δ 7.13 (s, 1H, aryl), 7.04 (d, 1H, aryl),
6.98 (d, 1H, aryl), 5.33 (t, 4H, olefinic), 4.12 (m, 2H, benzylic),
4.03 (t, 2H, OCH2), 3.99 (t, 2H, OCH2), 3.08 (m, 10H, 5 × CH2),
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CH2), 1.47 (m, 4H, 2 × CH2), 1.42-1.19 (m, 40H, 20 × CH2),
0.89 (t, 6H, CH3); 13C NMR (300 MHz, D2O) δ 149.74, 148.90,
129.62, 129.46, 123.92, 123.71, 69.36, 68.88, 51.12, 46.68, 38.99,
32.14, 29.97, 29.81, 29.60, 27.36, 26.53, 24.69, 24.22, 23.14, 23.06,
22.84, 14.05; HRMS (FAB): theory for C55H105N4O2 (M + 1),
853.8238; found (M + 1), 853.8264; Anal. (C55H109Cl4N4-
O2‚0.6H2O): C, H, N.
N-[4-(4-Amino-butylamino)-butyl]-3,4-bis-octadec-9-enyloxy-
benzamide, Dihydrochloride Salt (27). A concentrated solution
the amide 20 (0.22 g, 0.22 mmol) in ethyl acetate was added cooled
to 0 °C. A total of 5 mL of a freshly prepared saturated solution of
HCl in ethyl acetate was added dropwise and the solution stirred
for 1 h at room temperature during which time a white precipitate
formed. The ethyl acetate was removed in Vacuo and the residue
coevaporated with ethyl acetate and chloroform to give the product
27 as a white powder (0.19 g, 98%); 1H NMR (CDCl3) δ 8.95 (br
s, 2H, R2N+H2), 8.20 (br s, 3H, RN+H3), 7.45 (m, 2H, aryl), 6.78
(m, 1H, aryl), 5.33 (m, 4H, olefinic), 3.95 (m, 4H, OCH2), 3.38
(m, 2H, CH2), 3.11 (m, 2H, CH2), 3.01 (m, 4H, 2 × CH2), 2.08-
1.89 (m, 14H, 7 × CH2), 1.71 (m, 6H, 3 × CH2), 1.47-1.18 (m,
44H, 20 × CH2), 0.89 (t, 6H, CH3); HRMS (FAB): theory for
C51H94N3O3 (M + 1), 796.7290; found (M + 1), 796.7249; Anal.
(C51H95Cl2N3O3‚1.6H2O): C, H, N.
N-{4-[4-(4-Amino-butylamino)-butylamino]-butyl}-3,4-bis-oc-
tadec-9-enyloxy-benzamide, Trihydrochloride Salt (28). A con-
centrated solution of the amide 21 (0.22 g, 0.19 mmol) in ethyl

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