Defining the Molecular Requirements for the Selective Delivery of Polyamine Conjugates into Cells Containing Active Polyamine Transporters

Article abstract of DOI:10.1021/jm030223a

Several N¹-substituted polyamines containing various spacer units between nitrogen centers were synthesized as their respective HCl salts. The N¹-substituents included benzyl, naphthalen-1-ylmethyl, anthracen-9-ylmethyl, and pyren-1-

Full text of DOI:10.1021/jm030223a

J . Med. Chem. 2003, 46, 5129-5138
5129
Articles
Defin in g th e Molecu la r Requ ir em en ts for th e Selective Deliver y of P olya m in e
Con ju ga tes in to Cells Con ta in in g Active P olya m in e Tr a n sp or ter s
Chaojie Wang,† J ean-Guy Delcros,‡ Laura Cannon,§ Fanta Konate,§ Horacio Carias,§ J ohn Biggerstaff,§
Richard Andrew Gardner,§ and Otto Phanstiel IV*,§
Groupe de Recherche en Therapeutique Anticance´reuse, Faculte´ de Me´decine, 2, Avenue du Professeur Le´on Bernard,
University of Rennes 1, 35043 Rennes, France, and Department of Chemistry, P.O. Box 162366, University of Central Florida,
Orlando, Florida 32816-2366
Received May 13, 2003
Several N1-substituted polyamines containing various spacer units between nitrogen centers
were synthesized as their respective HCl salts. The N1-substituents included benzyl, naph-
thalen-1-ylmethyl, anthracen-9-ylmethyl, and pyren-1-ylmethyl. The polyamine spacer units
ranged from generic (4,4-triamine, 4,3-triamine, and diaminooctane) spacers to more exotic
[2-(ethoxy)ethanoxy-containing diamine, hydroxylated 4,3-triamine, and cyclohexylene-contain-
ing triamine] spacers. Two control compounds were also evaluated: N-(anthracen-9-ylmethyl)-
butylamine and N-(anthracen-9-ylmethyl)-butanediamine. Biological activities in L1210 (murine
leukemia), R-difluoromethylornithine (DFMO)-treated L1210, and Chinese hamster ovary (CHO)
and its polyamine transport-deficient mutant (CHO-MG) cell lines were investigated via IC50
cytotoxicity determinations. Ki values for spermidine uptake were also determined in L1210
cells. Of the series studied, the N1-benzyl-4,4-triamine system 6 had significantly higher IC50
values (lower cytotoxicity) in the L1210, CHO, and CHO-MG cell lines. A cellular debenzylation
process was observed in L1210 cells with 6 and generated “free” homospermidine. The size of
the N1-arylmethyl substituent had direct bearing on the observed cytotoxicity in CHO-MG cells.
The N1-naphthalenylmethyl, N1-anthracenylmethyl, and N1-pyrenylmethyl 4,4-triamines had
similar toxicity (IC50s: ∼0.5 µM) in CHO cells, which have an active polyamine transporter
(PAT). However, this series had IC50 values of >100 µM, 66.7 µM, and 15.5 µM, respectively,
in CHO-MG cells, which are PAT-deficient. The observed lower cytotoxicity in the PAT-deficient
CHO-MG cell line supported the premise that the conjugates use PAT for cellular entry. In
general, moderate affinities for the polyamine transporter were observed for the N-arylmethyl
4,4-triamine series with their L1210 Ki values all near 3 µM. In summary, the 4,4-triamine
motif was shown to facilitate entry of polyamine conjugates into cells containing active
polyamine transporters.
In tr od u ction
These range from neuroblastoma, melanoma, human
lymphocytic leukemia, colonic, and lung tumor cell lines
to murine L1210 cells.6a Because of the enhanced
cellular need for these amine growth factors and an
active transport system for their import, polyamine-
drug conjugates can be delivered to cancerous cell types.
This is possible due to the broad structural tolerance of
the PAT, which allows importation of non-native
polyamine constructs.
In vivo the native polyamines 1-3 exist as polycations
(as the nitrogens are protonated at physiological pH)
and are required for cell growth.1 Their alignment of
point charges are recognized by the polyamine transport
system and have been shown to facilitate their import.1-7
Rapidly dividing cells require large amounts of poly-
amines in order to grow. These can be internally
biosynthesized and also imported from exogenous sources.
Intracellular polyamine-production constraints in rap-
idly proliferating cells are thought to be partially offset
by scavenging polyamines from extracellular sources.6
In fact, many tumor types have been shown to contain
elevated polyamine levels and an active polyamine
transporter (PAT) for importing exogenous polyamines.6
Previous efforts have shown that specific cell types
can be targeted via the molecular recognition events
involved during the import of exogenous polyamines.1-31
Prior work in our laboratories evaluated the cytotoxicity
and polyamine transporter (PAT) affinity of conjugates
containing branched1,2 and linear polyamine motifs3,4
attached to either an anthracene or acridine nucleus.1,2,14
Indeed, certain linear triamines motifs were identified
as excellent vector systems.3,4 In particular, the 4,4-
triamine conjugate 4 (Figure 1, 4) had 150-fold higher
cytotoxicity in CHO cells (which contained an active
polyamine transport system) than in a mutant CHO-
* Corresponding author. Phone: (407) 823-5410. Fax: (407) 823-
2252. E-mail: ophansti@mail.ucf.edu.
† C.W. is a visiting scholar from the Department of Chemistry,
Henan University, Kaifeng, 475001, P.R. China.
‡ University of Rennes 1.
§ University of Central Florida.
10.1021/jm030223a CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/18/2003

5130 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Wang et al.
revealed the increased cytotoxicity (lower IC50 value) of
a N1-anthracenylmethyl conjugate over an N-acridinyl
analogue in murine leukemia (L1210) cells.1,2,4,14 In
addition, anthracene and the other polycyclic aromatic
systems provided a convenient UV “probe” for compound
identification and elicit a toxic response from cells upon
entry (presumably through DNA coordination).1-4,36
Therefore, a series of new arene conjugates was syn-
thesized (Figure 3, 6-15) and screened for uptake via
the polyamine transporter (PAT).
Their design served two purposes. First, 4 and 6-8
provided a homologous series, wherein a variety of
arenyl subunits were each attached to the “optimized”
4,4-triamine motif.3,4 Second, conjugates 9-14 provided
another series, wherein each member contained the
same N1-anthracenylmethyl subunit covalently attached
to a modified polyamine vector. By studying how these
materials influenced the survival of cells with active and
inactive polyamine transport systems, one can relate
how conjugate architecture influences cytotoxicity and
targeting of specific cell types. In short, by judicious
choice of amine substrates (e.g., a synthetic library) and
proper biological experiments (e.g., IC50, and Ki, meas-
urements) in selected cell types (murine leukemia
L1210, CHO and CHO-MG cells), a better understand-
ing between transporter affinity, cytotoxicity, and
polyamine-drug conjugate structure was obtained.
F igu r e 1. Native polyamines putrescine (PUT, 1), spermidine
(SPD, 2), spermine (SPM, 3) and anthracenyl-4,4-triamine
conjugate 4, R-difluoromethylornithine, (DFMO, 5a ) and L-
ornithine 5b.
MG cell line, which was PAT-deficient.3,4,32,33 Therefore,
cells which contain an active polyamine transport
system were much more sensitive to certain polyamine-
anthracene conjugates. The observed differential cyto-
toxicity formed the basis of a selective chemotherapeutic
strategy.
There are several caveats in using polyamines for
targeted drug delivery. First, rapidly dividing normal
cell types (e.g., bone marrow, intestinal epithelium, and
hair follicles) may also be effected by this strategy.
Whether polyamine conjugates can discriminate be-
tween cancer cell types and these other prolific cell lines
remains to be seen. Nevertheless, other authors have
observed that transformed cells are more sensitive than
normal cells to R-difluoromethylornithine (DFMO, 5a )
pretreatment.34,35 DFMO is a known ornithine decar-
boxylase (ODC) inhibitor. As shown in Figure 2, ODC
is responsible for putrescine biosynthesis and its inhibi-
tion has been shown to increase the uptake of extra-
cellular polyamines.34 Most pertinent to this study is
the observation that when DFMO is dosed in vivo, it
increased the uptake of radiolabeled putrescine specif-
ically into tumor cells, and not into other normal tissue,
even rapidly growing tissue.34 This suggests that DFMO
treatment may also increase the potency of the present
polyamine-conjugates. Second, the N1-component to be
delivered in this study (e.g., the arenylmethyl deriva-
tives) are relatively inert, and this conjugation strategy
may not be applicable to other drug classes. It is possible
that other anticancer agents, which have more “bio-
interactive” functional groups, would interact differently
with the PAT or other cell surface receptors and
diminish the anticipated selectivity. In this report, a
relatively inert architecture (e.g., an aryl unit) was used
to understand how changes in the N1-substituent and
polyamine sequence influenced drug potency and PAT
affinity.
Resu lts a n d Discu ssion
Syn th esis. While the terminally bis-alkylated poly-
amines have yielded diverse biological activity ranging
from anticancer to antidiarrheal agents,5,13 their mono-
substituted analogues have limited publications describ-
ing their use in vector design.1-5,14,17,21,23-31 This, in
part, may stem from their less direct syntheses, which
involve several steps.3-5,37-39 As shown in Scheme 1, the
reductive amination of 16c was achieved in two steps
via in situ generation of the imine 18. A series of amines
(17a -d ) was used to generate the crude imines 18a -
d . In general, solvent removal by rotary evaporation at
40-50 °C facilitated the conversion to 18, which was
then reduced to the respective secondary amine using
NaBH4 in excellent yield (∼85%). The amines were then
acidified with aqueous 4 N HCl to form the target HCl
salts: 10 (89%), 11 (81%), 14 (21%), and 15 (58%)3
(Scheme 1).
Compounds 12 and 13 were prepared using an amino-
alkanol strategy, which allowed for control of the amine
spacer group.3,4,37,39 As shown in Scheme 2, the reductive
amination of aldehydes 16a -d with 4-amino-1-butanol
and NaBH4 provided the substituted amino alcohols
19a -d . The tandem tert-butylcarbonylation and tosyl-
ation sequence resulted in the intermediates 20a -d ,
respectively. The tosylates 20a -d were generated as
The conjugates in this report (43 and 6-15) are
composed of an arene nucleus covalently bound to a
polyamine framework. The N1-arenylmethyl unit was
selected for alteration due to significant data, which
F igu r e 2. Polyamine biosynthetic pathway.

Delivery of Polyamine Conjugates into Cells
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5131
Sch em e 1
respective HCl salt with absolute ethanol. Compounds
4, 9, and 15 were generated in a prior report.3
Biologica l Eva lu a tion . Three cell lines were chosen
for bioassay. L1210 (mouse leukemia) cells were selected
to enable comparisons with the published IC50 and Ki
values known for a variety of polyamine substrates.5,29
Chinese hamster ovary (CHO) cells were chosen along
with a polyamine transport deficient mutant cell line
(CHO-MG) in order to comment on selective transport
via the PAT.3,4,14
Inhibition of ODC by DFMO (5a ) depletes intra-
cellular polyamine pools and leads to a significant
increase in polyamine uptake.34,35 Therefore, cells, which
are treated with DFMO, should be more susceptible to
the polyamine-vectored conjugates and should provide
lower IC50 values.3,4,6,14 IC50 values in L1210 cells with
and without DFMO-treatment were determined. Con-
jugates, which selectively target the PAT, should pro-
vide L1210/(L1210+DFMO) IC50 ratios greater than 1.
In fact, prior work in L1210 cells revealed an ap-
proximate doubling of potency of certain triamine
derivatives (50% reduction in IC50 value) with DFMO
treatment.
As shown in Table 1, a range of cytotoxicities was
observed, with 4 being the most cytotoxic and the
N-benzyl derivative 6 being the least efficacious. As
expected the controls 14 and 15 gave relatively low
cytotoxicity (and IC50 ratios <1) in both normal and
DFMO-treated L1210 cells (Table 1). Within the ho-
mologous series (4 and 6-8), the N1-naphthylmethyl 7,
N1-anthracenylmethyl 4, and N1-pyrenylmethyl 8 de-
rivatives had similar cytotoxicity both in the presence
and absence of DFMO. The dramatic reduction in
cytotoxicity for 6 was explained by the observation of
homospermidine (i.e., H2N(CH2)4NH(CH2)4NH2) within
cells treated with 6 (data not shown).
When DFMO-treated cells are depleted of putrescine
and spermidine, homospermidine can support cell
growth.40a A catabolic process whereby the N-benzyl
group was cleaved via cellular metabolism was observed
by the detection of homospermidine in the HPLC
analysis of perchloric acid extracts of cells treated with
6 using OPA postcolumn derivatization.40b Alterna-
tively, the detection of free homospermidine was also
facilitated by conversion to its dansyl derivative (see
F igu r e 3. Arene-polyamine conjugates.
crude mixtures and used immediately because they were
not stable to prolonged storage. Prior experience with
these materials revealed that it was not necessary to
isolate them. In fact higher yields were obtained, when
they were directly consumed in the subsequent step.3,4
As shown in Scheme 3, crude tosylate 20c was reacted
with either 1,3-diamino-2-hydroxypropane or trans-1,4-
diaminocyclohexane followed by 4 N HCl to give the
respective amino alcohol- and cyclohexyl-containing
triamines, 12 and 13 as their HCl salts.
In prior work, tosylate 20c was converted in two steps
to 4.3 Using a similar method shown in Scheme 4, the
crude tosylates 20a ,b,d (where a -d are defined in
Scheme 2) were reacted with excess putrescine 1 to form
adducts, which were treated with 4 N HCl to give the
desired triamines 6-8 as their trihydrochloride salts.
All the target compounds were purified by washing the

5132 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Wang et al.
Sch em e 2
Sch em e 3
was determined in a competitive assay with radiolabeled
spermidine.3,4,14,41 It should be noted that there are
numerous modes of entry for polyamines into cells and
that each mode may have a different structure-activity
relationship with our synthetic ligands. At present, we
were unable to discreetly measure each of these modes
of import. Therefore, the listed Ki values (Table 1) reveal
the overall transporter affinity of each synthetic conju-
gate when all these uptake modes are active in L1210
cells. These collective polyamine uptake modes will be
referred to as the “polyamine transporter” or PAT.
The affinities of substituted amine conjugates (4,
6-15) for the PAT in L1210 cells (i.e., Ki values) are
listed in Table 1. As expected the controls 14 and 15
gave high Ki values (low PAT affinity) and as mentioned
above no enhancement of toxicity with DFMO treat-
ment. This is consistent with earlier work which showed
that the innate affinity for the L1210 polyamine trans-
porter decreased with decreasing number of nitrogen
centers.4 In other words, tetraamines had higher PAT
affinity than triamines, triamines had higher affinity
than diamines, etc. Several of the triamine derivatives
(4, 6-9, 13) demonstrated moderate affinity for PAT (Ki
values < 6.2 µM). This feature was shown to be an
important parameter for facile import via the polyamine
transporter. Prior studies had shown that high-affinity
PAT ligands (like N-anthracenylmethyl-tetraamine con-
jugates) were not as potent cytotoxic agents as conju-
gates with moderate PAT affinity (e.g., related tri-
amines).4
Sch em e 4
Experimental Section) and measured via mass spectrom-
etry.40c,d Only trace quantities of homospermidine were
detected in L1210 cells treated with 7 and none with 4
or 8.
Similar findings were reported by Delcros and co-
workers, who demonstrated the catabolization of N-
benzylspermidine (PhCH2NH(CH2)3NH(CH2)4NH2) to
form free spermidine 2.40d Other N-dealkylations have
been reported by Bergeron et al. in their study of N1-
propylpolyamines.5 For example, the N1,N7-dipropyl-3,3-
triamine and N1,N8-dipropyl-4,4-triamine derivatives
were converted to their corresponding monopropyl
derivatives in 40% and 12%, respectively. The current
findings with 6, 7, 4, and 8 suggest a possible size
limitation for the N1 dealkylating enzyme in terms of
its inability to process the larger N1-substituents.
Previous studies revealed the similar IC50 values of
the Ant-(4,4)-triamine 4 and Ant-(4,3)-triamine 9 de-
rivatives in L1210 cells.3 Comparison of 9-12 provided
additional insights. Exchange of the internal nitrogen
with a CH2 group (10) or hydroxylation of the methylene
chain (12) gave higher IC50 values than found with the
parent system 9. Introduction of the polyether chain (11)
in lieu of the methylene chain present in 10 resulted in
a conjugate with lower toxicity and low affinity for the
PAT (Ki value: 90 µM). Not surprisingly, the cyclohexyl
derivative 13 had a similar profile as its parent 4 in
L1210 cells (with and without DFMO treatment). Over-
all, DFMO treatment gave little to no enhancement of
cytotoxicity for most systems studied with the exception
of 4 and 9.
Why are some analogues more cytotoxic than others?
One interpretation is that 4 is simply intrinsically more
toxic to the cell than the related controls 14 and 15. This
would assume that the polyamine architecture itself is
toxic to the cell and that different polyamine scaffolds
will have different cytotoxicities. Previous work, how-
ever, demonstrated that unsubstituted polyamines (i.e.
the 4,4-triamine) were not toxic to cells.3,4,40a A more
intriguing answer may lie in how efficiently these
materials recognize the PAT cell-surface receptor and
are transferred into the cell (or to their cellular target).
In light of the differential PAT affinities (Ki values)
observed with the unsubstituted polyamines,4 the latter
premise seems reasonable. In other words, the polyamine
“message” appears to be required for PAT recognition.
Changes in this message may also lead to differential
import rates (Vmax).4
An assessment of the conjugates’ ability to target the
PAT was conducted in two Chinese hamster ovary
(CHO) cell lines. The CHO-MG cell line is a polyamine-
transport deficient mutant, which was isolated for
growth resistance to MGBG, methylglyoxalbis(guanyl-
The Ki value is a measure of the affinity of the
polyamine conjugate for the polyamine transporter and

Delivery of Polyamine Conjugates into Cells
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5133
Ta ble 1. Biological Evaluation of Polyamine Derivatives in L1210 Cellsa
L1210
IC50 in µM
L1210+DFMO
IC50 in µM
L1210/(L1210+DFMO)
Ki values (µM)
L1210 cells
compd (tether)
4: Ant (4,4)
6: benzyl (4,4)
7: naphthyl (4,4)
8: pyrenyl (4,4)
IC50 ratioa
0.30 ((0.04)
36.3 (( 8.4)
0.50 ((0.03)
0.40 ((0.02)
0.4 ((0.1)
3.00 (( 0.07)
11.30 ((0.37)
1.50 ((0.08)
1.00 ((0.16)
6.30 ((0.26)
14.6 ((0.1)
0.15 (( 0.10)
421.0 (( 27.1)
0.43 (( 0.02)
0.36 (( 0.06)
0.20 (( 0.02)
4.60 (( 0.12)
17.00 (( 0.61)
2.30 (( 0.29)
0.7 (( 0.1)
2
1.8 ((0.1)
4.5 (( 0.8)
3.8 ((0.5)
2.9 ((0.3)
6.2 ((0.6)
13.3 ((1.5)
90.0 ((4.6)
12.5 ((1.2)
3.8 ((0.9)
32.2 ((4.3)
62.3 ((4.2)
0.1
1.1
1.1
2
0.7
0.7
0.7
1.4
0.7
0.7
9: Ant (4,3)
10: Ant-(octylene)
11: Ant-(diethoxy)
12: Ant- (4,3-hydroxyamino)
13: Ant-(cyclohexyl)
14: Ant-(butanediamine)
15: Ant (N-butyl)
9.78 (( 0.42)
21.9 ((3.6)
a
Note: the individual L1210 IC50 values are listed in µM and the ratio is dimensionless.
Ta ble 2. Biological Evaluation of Polyamine Derivatives in the CHO-MG and CHO Cell Lines
CHO-MG
CHO
(CHO-MG/CHO)
compound (tether)
4: Ant (4,4)
6: benzyl (4,4)
7: naphthyl (4,4)
8: pyrenyl (4,4)
IC50 in µM
IC50 in µM
IC50 ratio
66.7 (( 4.1)
>1000
>100
0.45 (( 0.10)
>1000
148
NA
>164
34
24
1
1.3
1
7
0.6 ((0.2)
0.46 ((0.05)
0.4 (( 0.1)
4.9 (( 0.2)
12.6 (( 0.6)
9.1 (( 0.4)
2.5 (( 0.5)
7.7 (( 0.5)
10.5 (( 2.0)
15.5 ((2.4)
9.5 ((1.1)
4.9 ((0.1)
15.9 ((1.5)
9.5 ((0.8)
17.4 ((2.8)
7.6 ((0.4)
11.2 ((2.3)
9: Ant (4,3)
10: Ant-(octylene)
11: Ant-(diethoxy)
12: Ant- (4,3-hydroxyamino)
13: Ant-(cyclohexyl)
14: Ant-(butanediamine)
15: Ant (N-butyl)
1
1.1
hydrazone), using a single-step selection after mutagen-
esis with ethyl methane sulfonate.32 MGBG is a known
substrate for PAT.32 For the purposes of this study, the
CHO-MG cell line represented cells with limited
polyamine transporter activity and provided a measure
of delivery independent of the PAT. In contrast, the
parent CHO cell line represented cell types with active
polyamine transport.32,33 Comparison of conjugate toxic-
ity in these two lines provided an important screen to
detect conjugate delivery via the PAT. For example, a
conjugate with high utilization of the transporter would
be very toxic to CHO cells, but less so to CHO-MG
cells.3,4,14
As shown in Table 2, the controls 14 and 15 gave the
same toxicity in both CHO and CHO-MG cells (CHO-
MG/CHO IC50 ratios near 1). The conjugates 10-12 also
gave similar IC50 values in both CHO cell lines. As
expected, this suggested that neither of these conjugates
were using the PAT to gain access to the cell and is
consistent with their corresponding low PAT affinity (as
observed in L1210 cells, with the respective Ki values
>12.5 µM). The N-benzyl derivative 6 was nontoxic to
both CHO cell lines. The remaining members of the N1-
aryl homologous series, N-naphthylmethyl 7, N-anthra-
cenylmethyl 4, and N-pyrenylmethyl 8, gave similar
cytotoxicities in CHO cells (IC50s ∼ 0.5 µM). In general,
the more hydrophobic the conjugate (e.g., 8), the more
cytotoxic it was to the PAT-deficient CHO-MG cells. For
example, the pyrene-containing conjugate 8 (ratio: 34)
gave a lower (CHO-MG/CHO) IC50 ratio than 4 (ratio:
148) and 7 (ratio: >164). In addition, the cyclohexyl
derivative 13 is more hydrophobic than 4 and was more
toxic (than 4) in CHO-MG cells. In fact 13 had similar
cytotoxicity as 8 in this cell line. This effect was likely
due to differential transport, wherein the hydrophobic
ligand accessed the cell via a non-PAT-mediated path-
way.
Evaluation of the triamine series in the two CHO cell
lines revealed striking preferences by certain polyamine
architectures for the PAT. Prior findings suggested the
4,4-triamine 4 as an excellent vector as its anthracene
conjugate displayed a nearly 150-fold higher toxicity in
CHO cells over the CHO-MG cell line.13 However, this
study revealed that the selective targeting of cells with
active polyamine transporters can be further enhanced
via the smaller, less hydrophobic naphthylmethyl unit
in 7. Of the series tested, the naphthylmethyl triamine
7 gave the best selectivity profile. In short, 7 had
relatively low toxicity in cells with low PAT activity and
high toxicity in cells with high polyamine transport
activity. Moreover, our findings reiterate that CHO and
CHO-MG IC50 comparisons are an excellent way to rank
polyamine vectors and their transport.14 As before3, the
cytotoxicity of the triamines in the CHO lines correlated
nicely with the L1210 IC50 and Ki results.
A priori one may have expected that polyamines with
bulky N-substituents would be poor ligands for the
polyamine transporter. However, earlier comparisons
suggested that the bulky N-(arylmethyl) substituent
actually enhances the PAT binding affinity and cyto-
toxicity of the polyamine conjugate in vitro.4 Although
aromatic substituents seem to impart higher affinity for
cell-surface receptors, there are limitations. As seen
with 8 if the arene unit is too hydrophobic then the
selectivity for the polyamine transporter is diminished
(Table 2). Using deconvolution microscopy, conjugates
such as 4 with high cytotoxicity had a high qualitative
rate of import. By comparison a related anthracene
tetraamine conjugate with lower cytotoxicity was less
internalized during the same time period. These pre-
liminary findings suggested that the differential cyto-
toxicities could be explained by different rates of trans-
port into the cell.4 Collectively, these observations
further support the ability of certain cell types to

5134 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Wang et al.
F igu r e 4. Deconvolution microscopy images of detached A375 cells treated with 4 (Panels 1-3). Panel 1 (3 min),4 Panel 2 (10
min), and Panel 3 (60 min) represent cells incubated with 4, for the respective time period, at 10 µM, then washed with buffer
and fixed with paraformaldehyde.
recognize the conjugates via their polyamine motifs and
N1-substituents.
compartment via a cationic symporter.30 Indeed, panel
1 in Figure 4, seems to have captured this endocytosis
event in action. Using the same model, to exit the vesicle
(via the putative cationic symporter), the conjugate must
dissociate from its vesicular PAT receptor. Since tri-
amines have moderate binding affinities they should be
able to dissociate from the internalized PAT receptor
to exert their toxic effect. Panel 2 (at 10 min) revealed
an increase in the number and size of the internalized
vesicles. Inspection of Panel 3 (at 60 min) in Figure 4
suggests that at latter times the conjugate 4 has escaped
the initially formed vesicles seen in Panel 1 and is
delivered throughout the cell. To the best of our knowl-
edge, this is the first time-course study of a polyamine
import process using a fluorescent, bioactive-marker.
A polyamine transporter gene has not yet been
identified in mammalian cells. In fact, little is known
about the structural aspects of the polyamine trans-
porter (PAT) itself. Proteoglycans may play a role in
polyamine transport.44 Proteoglycans are composed of
glycosaminoglycan chains covalently linked to protein
and expression of cell-surface proteoglycans are required
for the binding of many extracellular substrates.44c
Indeed, inhibition of polyamine biosynthesis with DFMO
resulted in an increase of cell-associated proteoglycans
exhibiting higher affinity for spermine in a human lung
fibroblast study.44c The data indicated a specific role for
heparan sulfate proteoglycans (HSPG) in the uptake of
spermine by fibroblasts. These provocative results sug-
gest that proteoglycan-mediated polyamine transport
may play an important role in future drug delivery
strategies.45,46 The current report also provides new
fluorescent probes needed to evaluate these interesting
new pathways.
To further address this issue, we conducted a time-
course microscopy study to observe the delivery profile
of 4 into A375 melanoma cells. Melanoma cells were
chosen due to their large size and susceptibility to
polyamine analogues.4,6a A375 cells were incubated with
conjugate 4 (at 10 µM) for 3, 10, and 60 min time
intervals. A series of optical “slices” of the resultant cells
were obtained, and one slice was chosen to best repre-
sent the sample overall. The UV spectra of the an-
thracene conjugates suggest a consistent λmax near 364
nm and an emission maxima near 417 nm. Therefore,
the anthracene component was easily tracked via its
diagnostic fluorescence properties and selected images
from the time-course study are shown in white/gold in
Figure 4. The relative levels of fluorescence inside the
cell (at early and late times) confirmed the expected
time-dependent transport phenomenon. Panel 1 in
Figure 4 revealed that the triamine conjugate 4 rapidly
accessed the cell as evidenced by the large number of
fluorescent vesicles inside the cell after a three minute
exposure of cells to 4.4
The samples were washed before they were fixed with
paraformaldehyde and imaged by deconvolution micros-
copy. Prior studies showed that both the surface-bound
and unbound triamine were efficiently washed away
from the cell surface in the workup step.4 The limited
amount of triamine bound to the cell surface is con-
sistent with the lower binding affinity of this probe to
PAT (Table 2) and presumably other membrane recep-
tors.4 Conversely, high affinity tetraamine ligands (e.g.,
L1210 Ki values in the nM range) were shown to bind
irreversibly to the membrane and gave large amounts
of conjugate still bound to the cell surface after a buffer
wash.4 The lower cytotoxicity of the higher affinity
tetraamine systems was rationalized by their indis-
criminate binding to numerous cell surface receptors
which likely result in dramatic changes in membrane
fluidity and transport (e.g., endocytosis).4
In summary, the polyamine transporter is capable of
distinguishing between systems, which have the same
overall length, but a different separation of point
charges and N1-substituents.3,4,26-29 The current study
identified hydrophobicity (and possible size) limitations
of the appended arenyl unit, which influence the selec-
tivity profile of the polyamine conjugate. Conjugates
containing the presence of a 4,4-triamine sequence,
moderate PAT affinity (Ki values near 1 µM), moderate
hydrophobicity (e.g., 4 or 7), and high (CHO-MG/CHO)
IC50 ratios were shown to target cells via the PAT
pathway with high specificity.
The findings were also consistent with a model
proposed by Cullis and Poulin.6a,30,43 First, polyamines
bind to a surface receptor, which results in membrane
envagination (endocytosis) to create polyamine-rich
vesicles inside the cell. The derivative may then be
exported out of this vesicle to a particular cellular

Delivery of Polyamine Conjugates into Cells
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5135
Triton in distilled water. Two hundred microliters of the cell
lysate was transferred in a 5 mL scintillation vial containing
3 mL of Pico-Fluor15. The respective radioactivity of each
sample was measured using a scintillation counter. The Km
value of spermidine transport was determined by Lineweaver-
Burke analysis as described.48
The ability of conjugates to compete for [14C]spermidine
uptake were determined in L1210 cells by a 10-min uptake
assay in the presence of increasing concentrations of competi-
tor, using 1 µM [14C]spermidine as substrate. Ki values for
inhibition of spermidine uptake were determined using the
Cheng-Prusoff equation41 from the IC50 value derived by
iterative curve fitting of the sigmoidal equation describing the
velocity of spermidine uptake in the presence of the respective
competitor.43,49 Cell lines (murine leukemic L1210 cells) were
grown and maintained according to established procedures.7,42
Cells were washed twice in HBSS prior to the transport assay.
Con clu sion s
A series of N1-substituted polyamines containing
different spacer units between nitrogen centers was
synthesized. DFMO-treated L1210 cells were not overly
sensitive to most of the conjugates evaluated with the
exception of 4 and 9. A survey of different polyamine
vectors revealed that the respective 4,4-triamine system
had high selectivity for CHO cells containing active
polyamine transporters. Optimal use of this transporter
seems to require a balance between PAT binding affin-
ity, conjugate internalization via vesicles, and escape
to the cellular target.
In summary, selective vector motifs were identified,
which enable cellular entry via the polyamine trans-
porter (PAT). Since many cancer cell lines have high
PAT activity, especially when dosed with DFMO,34 these
results further illustrate a novel anticancer strategy.
Decon volu tion Micr oscop y. The microscope system in-
cluded an Applied Precision (Issaquah, WA) Deltavision
system equipped with a Nikon Eclipse TE200 inverted micro-
scope. Image deconvolution was performed using Applied
Precision SoftWorX software. Detached melanoma A375 cells3
were incubated with 4 (10 µM) at 37 °C for the respective time
interval in a culture medium containing 10% fetal bovine
serum in RPMI-1640 media containing phenol red and an
antibiotic cocktail. The cells were then washed with fresh
phosphate-buffered saline (PBS) and centrifuged, and the
supernatant was removed. The cellular pellet was resuspended
in fresh PBS, fixed with paraformaldehyde, and placed onto a
microscope slide and imaged by the deconvolution microscope.
This technique provides an image of the entire cell by using
software to overlay a series of planar images (or slices) to give
a stacked translucent view of the entire cell. The lightest
regions in Figure 4 contain the highest concentration of the
fluorescent conjugate.
Exp er im en ta l Section
Ma t er ia ls. 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. TLC solvent systems are based on volume %, and
NH4OH refers to concentrated aqueous NH4OH. Elemental
analyses were performed by Atlantic Microlabs (Norcross, GA).
High-resolution mass spectrometry was performed by Dr.
David Powell at the University of Florida Mass Spectrometry
facility.
Biologica l Stu d ies. Murine leukemia cells (L1210), CHO,
and CHO-MG cells were grown in RPMI medium (Eurobio,
Les Ulis, France) supplemented with 10% fetal calf serum, 2
mM glutamine (2 mM), penicillin (100U/mL), and streptomycin
(50 µg/mL) (BioMerieux, Marcyl’Etoile, France). L-Proline (2
µg/mL) was added to the culture medium for CHO-MG cells.
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. Typically, samples
contain less than 5% trypan blue positive cells (dead). For
example, L1210 cells in early to mid log-phase were used.
IC50 Deter m in a tion s. Cell growth was assayed in sterile
96-well microtiter plates (Becton-Dickinson, Oxnard, CA).
L1210 cells were seeded at 5 × 104 cells/mL of medium (100
µL/well). Single CHO and CHO-MG cells, harvested by
trypsinization were plated at 2 × 103 cells/mL. Drug solutions
(5 µL per well) of appropriate concentration were added at the
time of seeding for L1210 cells and after an overnight incuba-
tion for CHO and CHO-MG cells. In some experiments DFMO
(5 mM) was added in the culture medium at the time of drug
addition. 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 (Labsystems, Cergy-
Pontoise, France) for absorbance (540 nm) measurements.47
Ki P r oced u r e. The ability of the conjugates to interact with
the polyamine transport system were determined by measur-
ing competition by the conjugates against radiolabeled sper-
midine uptake in L1210 cells. This procedure was used to
obtain the data listed in Table 1. Initially, the Km value of
spermidine transport was determined in a reaction volume of
600 µL of Hanks’s balanced salt solution (HBSS) containing 3
× 106 cells/mL in the presence of 0.5, 1, 2, 4, 6, and 8 µM [14C]-
spermidine. The cell suspensions were incubated at 37 °C for
10 min. The reaction was stopped by adding 3 mL ice-cold
phosphate-buffered saline (PBS). The tubes were centrifuged,
and the supernatant was removed. The cell pellets were then
washed twice with ice-cold PBS, and the supernatant was
removed. The pellet was broken by sonication in 500 µL of 0.1%
Gen er a l P r oced u r e for Im in e F or m a tion . To a stirred
solution of amine derivative (12 mmol) in 25% MeOH/CH2Cl2
(10 mL), was added a solution of 9-anthraldehyde (10 mmol)
in 25% MeOH/CH2Cl2 (10 mL) under N2. The mixture was
stirred at room-temperature overnight until the imine forma-
tion was complete (monitored by TLC). The solvent was
evaporated under vacuum to give the crude imine 18 as a
bright green solid, which was used for reduction without
further purification.
Gen er a l P r oced u r e for t h e R ed u ct ion of Im in es.
NaBH4 (30 mmol) was added in small portions to the solution
of crude imine in 1:1 CH3OH :CH2Cl2 (20 mL) at 0 °C. The
mixture was stirred at room-temperature overnight, then
concentrated under vacuum. The residue was dissolved in
CH2Cl2 (50 mL) and washed three times with 50 mL of aqueous
Na2CO3 (pH ) 10). The organic layer was separated, dried over
anhydrous Na2SO4, filtered, and concentrated under vacuum.
The residue was purified by flash chromatography on silica
gel to provide the secondary amines, which were immediately
converted to their HCl salts (see General Procedure for the
HCl Salt Formation).
Gen er a l P r oced u r e for N-Boc P r otection a n d Tosyl-
ation . Note: During the synthesis of starting N1-BOC diamino-
octane (17a ) the molar ratio of 1,8-diaminooctane to Boc2O was
3:1. The other BOC reactions were performed in the following
molar ratios.
N-Boc In tr od u ction . As an example, a solution of the
respective secondary amine 19 (5 mmol) in triethylamine-
methanol (1:9 V/V, 20 mL) was stirred at 0 °C for 10 min, then
a solution of di-tert -butyl dicarbonate (7.5 mmol) in methanol
(5 mL) was added dropwise over 10 min. The mixture was
stirred for an additional 1 h at 0 °C. The temperature was
allowed to gradually rise to room temperature, and the
reaction was stirred overnight. The mixture was evaporated
under reduced pressure. The residue was dissolved in CH2Cl2
and washed with deionized water several times. The organic
layer was separated, dried with anhydrous Na2SO4, filtered,
and concentrated. The residue could be purified by flash

5136 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Wang et al.
column chromatography on silica gel (to give g84% yield)3 or
be used in the next step without further purification.
O-Tosyla tion (P r ep a r a tion of 20). A solution of the N-Boc
protected alcohol (5 mmol) in dry pyridine (20 mL) was stirred
at 0 °C for 10 min. p-Toluenesulfonyl chloride (TsCl, 7.5 mmol)
was added in small portions over 30 min. The mixture was
stirred for an additional 1 h at 0 °C, then the reaction flask
was placed in a refrigerator (0-5 °C) overnight. The mixture
was poured into 200 mL of ice-water, and a viscous liquid
typically precipitated. After the upper water layer was de-
canted, the remaining viscous liquid was dissolved in meth-
ylene chloride and washed with deionized water several times.
These steps avoided an emulsion during the extraction workup.
The organic layer was separated, dried with anhydrous
Na2SO4, filtered, and concentrated. The residue could be
purified by flash column chromatography on silica gel (to give
50-88% yields)3 or be used in the next step without further
purification.3,4
Gen er a l P r oced u r e for th e Su bstitu tion of th e Tosyl-
a ted Com p ou n d s w ith Differ en t Dia m in es. The crude
tosylate 20 (1 mmol) and the respective diamine (5 mmol) were
dissolved in acetonitrile (10 mL) and stirred at 75 °C under
N2 overnight. After disappearance of the tosylate was con-
firmed by TLC, the solution was concentrated under reduced
pressure. The residue was dissolved in CH2Cl2 (20 mL) and
washed three times with saturated aqueous sodium carbonate.
The organic layer was separated, dried with anhydrous sodium
sulfate, filtered, and concentrated under vacuum. The residue
was purified by flash column chromatography on silica gel.
The purified products were isolated in good yields (60-75%)
and converted to their HCl salts as described below.
Gen er al P r ocedu r e for th e HCl Salt For m ation (P r epa-
r a tion of 4 a n d 6-15). The respective amine or N-Boc
protected amine (0.5 mmol) was dissolved in EtOH (5 mL) and
stirred at 0 °C for 10 min. 4 N HCl (8 mL) was added dropwise
at 0 °C. The reaction mixture was stirred at room-temperature
overnight. The solution was concentrated under reduced
pressure below 40 °C and typically gave a bright yellow solid
as a precipitate. The solids were washed several times with
absolute ethanol and dried under vacuum to give the pure
target compounds (4 and 6-15). The yields were typically
>70%.
2H), 1.63 (m, 2H), 1.42 (br s, 8H); 13C NMR (D2O): δ 130.6,
130.3, 130.0, 129.4, 127.6, 125.5, 122.4, 120.5, 47.8, 42.3, 39.7,
28.2, 28.1, 26.9, 25.9, 25.7, 25.4; HRMS (FAB) m/z calcd for
C
23H31N2 (M + H - 2HCl)+: 335.2487; found: 335.2489. Anal.
(C23H32Cl2N2) C, H, N.
{8-[(An th r acen -9-ylm eth yl)-am in o]-octyl}-car bam ic acid
ter t-bu tyl Ester (10a ). The secondary amine precursor to 10
was also isolated after NaBH4 reduction. 10a : yellow viscous
liquid; yield 84%; Rf ) 0.24, methanol/chloroform, 1:99; 1H
NMR (300 MHz, CDCl3) δ 8.40 (s, 1H), 8.36 (d, 2H), 8.00 (d,
2H), 7.53 (t, 2H), 7.46 (t, 2H), 4.72 (s, 2H), 4.50 (br s, 1H,
BocNH), 3.08 (m, 2H), 2.84 (t, 2H), 1.60 (m, 2H), 1.42 (br s,
10H), 1.28 (br s, 9H); 13C NMR: δ 155.7, 131.6, 131.3, 130.0,
128.9, 126.9, 125.9, 124.7, 123.9, 78.9, 50.5, 45.7, 40.6, 30.0
(2C), 29.4, 29.2, 28.4, 27.3, 26.7; HRMS (FAB) m/z calcd for
C
28H39N2O2 (M + H)+: 435.3012; found: 435.3048. Anal.
(C28H38N2O2‚0.15H2O) C, H, N.
2-(2-{2-[(An th r acen -9-ylm eth yl)-am in o]-eth oxy}-eth oxy)-
eth yla m in e Dih yd r och lor id e (11). Bright yellow solid; yield
81%; 1H NMR (300 MHz, DMSO-d6 +D2O) δ 8.80 (s, 1H), 8.40
(d, 2H), 8.20 (d, 2H), 7.68 (t, 2H), 7.60 (t, 2H), 5.24 (s, 2H),
3.82 (t, 2H), 3.66 (m, 4H), 3.60 (t, 2H), 3.40 (t, 2H), 2.96 (t,
2H); 13C NMR(D2O): δ 130.8, 130.5, 130.1, 129.5, 127.7, 125.6,
122.5, 120.5, 69.8 (2C), 66.6, 65.1, 47.0, 42.5, 39.2. HRMS
(FAB) m/z calcd for: C21H27N2O2 (M + H-2HCl)+: 339.2073;
found: 339.2074. Anal. (C21H28Cl2N2O2) C, H, N.
1-Am in o-3-{4-[(a n t h r a cen -9-ylm et h yl)-a m in o]-b u t yl-
a m in o}-p r op a n -2-ol Tr ih yd r och lor id e (12). Bright yellow
solid, yield 95%. 1H NMR (DMSO-d6+D2O): δ 8.80 (s, 1H),
8.42 (d, 2H), 8.20 (d, 2H), 7.68 (t, 2H), 7.60 (t, 2H), 5.22 (s,
2H), 4.20(m, 1H), 3.28 (t, 2H), 3.1∼2.80 (m, 6H), 1.78 (brs, 4H).
13C NMR (D2O): δ 130.6, 130.4, 130.0, 129.5, 127.7, 125.5,
122.5, 120.3, 63.8, 50.1, 47.3, 47.2, 42.8, 42.4, 23.0, 22.9. HRMS
(FAB): calcd for
C
22H30N3O (M + H-3HCl)+: 352.2389;
found: 352.2381. Anal. (C22H32Cl3N3O‚0.4H2O) C, H, N.
N-{4-[(An th r a cen -9-ylm eth yl)-a m in o]-bu tyl}-cycloh ex-
a n e-1,4-d ia m in e Tr ih yd r och lor id e (13) . Bright yellow
solid, yield 95%. 1H NMR (CD3OD): δ 8.68 (s, 1H), 8.41 (d,
2H), 8.17 (d, 2H), 7.68 (t, 2H), 7.57 (t, 2H), 5.34 (s, 2H), 3.40
(m, 2H), 3.16 (m, 4H), 2.26(m, 2H), 2.20(m, 2H), 1.90(m, 4H),
1.60(m, 4H); 13C NMR(D2O): δ 130.7, 130.4, 130.1, 129.5,
127.7, 125.5, 122.5, 120.3, 55.4, 48.7, 47.1, 44.3, 42.8, 28.2, 26.8,
23.3, 23.0. HRMS (FAB): calcd for C25H34N3 (M + H-3HCl)+:
376.2753; found: 376.2747. Anal. (C25H36Cl3N3‚1.0H2O) C, H,
N.
Note: Compounds 4, 9, and 15 were synthesized in a
previous report.3
N-(4-Am in o-bu tyl)-N′-ben zen e-1-ylm eth yl-bu ta n e-1,4-
N1-An t h r a cen -9-ylm et h yl-b u t a n e-1,4-d ia m in e Dih y-
dr och lor ide (14). Yellow solid; yield 21%; Rf ) 0.11, methanol/
chloroform, 1:20 + 3 drops of NH4OH); 1H NMR (300 MHz,
D2O): δ 8.55 (br s, 1H), 8.15 (d, 2H), 8.08 (d, 2H), 7.66 (m,
1
d ia m in e Tr ih yd r och lor id e (6). White solid; yield 73%. H
NMR (300 MHz, DMSO+D2O): δ 7.47 (br s, 2H), 7.41 (br s,
3H), 4.09 (s, 2H), 2.88 (br s, 6H), 2.78 (br s, 2H), 1.63 (br s,
8H). 13C NMR (D2O): 130.7, 129.9 (2C), 129.8, 129.4 (2C), 51.3,
47.13, 47.07, 46.5, 39.0, 24.2, 23.1, 23.0 (2C); HRMS (FAB):
calcd for C15H28N3 (M + H - 3HCl)+: 250.2283; found:
250.2268. Anal. (C15H30Cl3N3) C, H, N.
4H), 5.08 (br s, 2H), 3.25 (t, 2H), 3.0 (t, 2H), 1.76 (m, 4H); 13
C
NMR (D2O): δ 130.6, 130.3, 129.9, 129.4, 127.6, 125.4, 122.4,
120.2, 47.2, 42.7, 38.6, 24.2, 22.9; ESI-MS m/z calcd for
C
19H23N2 (M + H): 279.2; found: 279.2. Anal. (C19H24N2Cl2,‚
N-(4-Am in o-bu tyl)-N′-n a p h th a len -1-ylm eth yl-bu ta n e-
1,4-d ia m in e Tr ih yd r och lor id e (7). White solid; yield 80%.
1H NMR (300 MHz, D2O): δ 8.03 (brs, 1H), 7.96 (brs, 2H), 7.59
(brs, 2H), 7.53 (brs, 1H), 7.51 (brs, 1H), 4.68 (s, 2H), 3.14 (t,
2H), 2.99 (m, 6H), 1.67 (brs, 8). 13C NMR (D2O): 132.9, 130.2,
129.9, 128.8, 128.5, 126.8, 126.0, 125.8, 125.0, 121.9, 47.4, 46.5,
46.4, 46.3, 38.4, 23.5, 22.5, 22.4 (2C); HRMS(FAB): calcd for
0.2H2O) C, H, N.
N1-An th r a cen -9-ylm eth yl-bu tyla m in e m on oh yd r och lo-
r id e (15) was synthesized in a previous report.3
(8-Am in o-octyl)-ca r ba m ic Acid ter t-bu tyl Ester (17a ).
white solid; yield 78%; Rf ) 0.33, methanol/chloroform, 1:9;
1H NMR (300 MHz, CDCl3) δ 4.50 (br s, 1H, BocNH), 3.18 (m,
2H), 2.65 (t, 2H), 1.42 (br s, 12H), 1.36 (br s, 9H); 13C NMR:
δ 155.7, 78.9, 42.1, 40.5, 33.7, 30.0, 29.3, 29.2, 28.4 (3C), 26.8,
26.7; HRMS (FAB) m/z calcd for C13H29N2O2 (M + H)+:
245.2229; found: 245.2235.
4-(N-Ben zyla m in o)-bu ta n -1-ol (19a ). Pale yellow liquid;
yield 89%; Rf ) 0.34, methanol/chloroform, 1:4); 1H NMR (300
MHz, CDCl3) δ 7.29 (brs, 5H), 3.77 (s, 2H), 3.59 (t, 2H), 2.68
(t, 2H), 1.65 (brs, 4H); 13C NMR: δ 138.8, 128.4 (2C), 128.1
(2C), 127.1, 62.5, 53.7, 49.1, 32.4, 28.5; HRMS (FAB) m/z calcd
for C11H18NO(M + H)+: 180.1388; found: 180.1389. Anal.
(C11H17NO‚0.25H2O) C, H, N.
C
19H30N3 (M + H - 3HCl)+: 300.2440; found: 300.2431. Anal.
(C19H32Cl3N3‚0.57H2O) C, H, N.
N-(4-Am in o-bu tyl)-N′-p yr en -1-ylm eth yl-bu ta n e-1,4-d i-
a m in e Tr ih yd r och lor id e (8). White solid; yield 94%. 1H
NMR (300 MHz, D2O): δ 8.1∼7.6 (m, 9H), 4.46 (s, 2H), 3.04
(m, 2H), 2.92 (m, 6H), 1.63 (br s, 8H). 13C NMR (D2O): δ 131.3,
130.4, 129.7, 128.4, 128.2, 127.9, 126.9, 126.3, 125.7, 125.6,
124.53, 124.51, 123.1, 122.9, 122.5, 120.9, 47.9, 47.0, 46.9, 46.6,
39.0, 24.1, 23.0, 22.9 (2C). HRMS (FAB): calcd for C25H31N3
(M + H - 3HCl)+: 374.2596; found: 374.2594. Anal. (C25H34
Cl3N3‚1.2H2O) C, H, N.
-
N1-An th r a cen -9-ylm eth yl-octa n e-1,8-d ia m in e Dih yd r o-
ch lor id e (10). Yellow solid; yield 89%; 1H NMR (300 MHz,
CD3OD) δ 8.69 (s, 1H), 8.40 (d, 2H), 8.15 (d, 2H), 7.70 (t, 2H),
7.59 (t, 2H), 5.32 (s, 2H), 3.30 (br s, 2H), 2.92 (t 2H), 1.82 (m,
N-(Na p h th a len -1-ylm eth yl)-4-a m in o-bu ta n -1-ol (19b).
Dark yellow liquid; yield 93.6%; Rf ) 0.54, methanol/
chloroform, 1:4); 1H NMR (300 MHz, CDCl3) δ 8.02 (d, 1H),
7.85 (d, 1H), 7.76 (d, 1H), 7.47 (m, 4H), 4.23 (s, 2H), 3.58 (t,

Delivery of Polyamine Conjugates into Cells
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5137
2H), 2.79 (t, 2H), 2.51 (brs, 2H), 1.68 (brs, 4H); 13C NMR: δ
(13) (a) Bergeron, R. J .; Wiegand, J .; McManis, J . S.; Weimar, W.
R.; Smith, R. E.; Algee, S. E.; Fannin, T. L.; Slusher, M. A.;
Snyder, P. S. Polyamine Analogue Antidiarrheals: A Structure-
Activity Study. J . Med. Chem. 2001, 44, 232-244. (b) Bergeron,
R. J .; Yao, G. W.; Yao, H.; Weimar, W. R.; Sninsky, C. A.; Raisler,
B.; Feng, Y.; Wu, Q.; Gao, F. “Metabolically Programmed
Polyamine Analogue Antidiarrheals. J . Med. Chem. 1996, 39,
2461-2471. (c) Bergeron, R. J .; Neims, A. H.; McManis, J . S.;
Hawthorne, T. R.; Vinson, J . R. T.; Bortell, R.; Ingeno, M. J .
Synthetic polyamine analogues as antineoplastics. J . Med. Chem.
1988, 31, 1183-1190. (d) Casero, R. A., J r.; Woster, P. M.
Terminally Alkylated Polyamine Analogues as Chemotherapeu-
tic Agents. J . Med. Chem. 2001, 44, 1-26 and references therein.
(14) Delcros, J .-G.; Tomasi, S.; Carrington, S.; Martin, B.; Renault,
J .; Blagbrough, I. S.; Uriac, P. Effect of spermine conjugation
on the cytotoxicity and cellular transport of acridine. J . Med.
Chem. 2002, 45, 5098-5111.
(15) Azzam, T.; Eliyahu, H.; Shapira, L.; Linial, M.; Barenholz, Y.;
Domb, A. J . Polysaccharide-Oligoamine Based Conjugates for
Gene Delivery. J . Med. Chem. 2002, 45, 1817-1824.
(16) Stark, P. A.; Thrall, B. D. Meadows, G. G.; Abdul-Monem, M.
M. Synthesis and Evaluation of novel spermidine derivatives as
targeted cancer chemotherapeutic agents. J . Med. Chem. 1992,
35, 4264-4269.
134.5, 133.6, 131.3, 128.6, 127.8, 126.2, 126.1, 125.5, 125.2,
122.9, 62.4, 51.0, 49.6, 32.1, 28.3; HRMS m/z calcd for C15H19
-
NO(M+): 229.1467; found: 229.1477. Anal. (C15H19NO‚0.2H2O)
C, H, N.
4-[(An t h r a cen -9-ylm et h yl)-a m in o]-b u t a n -1-ol (19c).3
Bright yellow solid; yield 81%; Rf ) 0.51, methanol/chloroform,
1
1:9; H NMR (300 MHz, CDCl3): δ 8.40 (s, 1H), 8.26 (d, 2H),
8.00 (d, 2H), 7.49 (m, 4H), 4.68 (s, 2H), 3.50 (t, 2H), 2.90 (t,
2H), 1.65 (br s, 4H); 13C NMR: δ 131.7, 130.7, 130.5, 129.5,
127.8, 126.6, 125.3, 124.0, 62.9, 50.6, 45.7, 32.7, 29.1. HRMS
(FAB) m/z calcd for: C19H22NO (M + H)+: 280.1701; found:
280.1679. Anal. (C19H21NO) C, H, N.
4-[(P yr en -1-ylm eth yl)-a m in o]-bu ta n -1-ol (19d ). White
1
solid; yield 94%. H NMR (300 MHz, CDCl3): δ 8.24 (d, 2H),
8.13 (m, 4H), 7.96 (m, 4H), 4.42 (s, 2H), 3.56 (t, 2H), 2.82 (t,
2H), 1.64 (brs, 4H). 13C NMR (CDCl3): 132.4, 131.0, 130.6,
130.5, 128.7, 127.7, 127.2, 127.0 (2C), 125.7, 125.0, 124.9,
124.8, 124.6 (2C), 122.4, 62.6, 51.3, 49.6, 32.3, 28.6. HRMS
(FAB): calcd for
304.1701. Anal. (C21H21NO‚0.2H2O) C, H, N.
C
21H22NO(M + H)+: 304.1701; found:
(17) Cohen, G. M.; Cullis, P.; Hartley, J . A.; Mather, A. Symons, M.
C. R.; Wheelhouse, R. T. Targeting of Cytotoxic Agents by
Polyamines: Synthesis of a Chloroambucil-Spermidine Conju-
gate. J . Chem. Soc., Chem. Commun. 1992, 298-300.
(18) Cai, J .; Soloway, A. H. Synthesis of Carboranyl Polyamines for
DNA Targeting. Tetrahedron Lett. 1996, 37, 9283-9286.
(19) Ghaneolhosseini, H.; Tjarks, W.; Sjoberg, S. Synthesis of Novel
Boronated Acridines and Spermidines as Possible Agents for
BNCT. Tetrahedron 1998, 54, 3877-3884.
Ack n ow led gm en t. This work was supported in part
by the Elsa U. Pardee Foundation and the Florida
Hospital Gala Endowed Program for Oncologic Re-
search. The authors wish to thank the Chinese Scholar-
ship Council for partial support of C.W.
(20) Blagbrough, I. S.; Geall, A. J . Homologation of Polyamines in
the Synthesis of Lipo-Spermine Conjugates and Related Lipo-
plexes. Tetrahedron Lett. 1998, 443-446.
(21) Cullis, P. M.; Merson-Davies, L.; Weaver, R. Conjugation of a
polyamine to the bifunctional alkylating agent chlorambucil does
not alter the preferred cross-linking site in duplex DNA. J . Am.
Chem. Soc. 1995, 117, 8033-8034.
(22) Aziz, S. M.; Yatin, M.; Worthen, D. R.; Lipke, D. W.; Crooks, P.
A. A novel technique for visualising the intracellular localization
and distribution of transported polyamines in cultured pulmo-
nary artery smooth muscle cells. J . Pharm. Biomed. Anal. 1998,
17, 307-320.
(23) Saab, N. H.; West, E. E.; Bieszk, N. C.; Preuss, C. V.; Mank, A.
R.; Casero, R. A.; Woster, P. M. Synthesis and Evaluation of
Polyamine Analogues as Inhibitors of Spermidine/Spermine-N1-
Acetyltransferase (SSAT) and as Potential Antitumor agents.
J . Med. Chem. 1993, 36, 2998-3004.
(24) Porter, C. W.; Cavanaugh, P. F.; Ganis, B.; Kelly, E.; Bergeron,
R. J . Biological properties of N-4 and N-1, N-8-spermidine
derivatives in cultured L1210 leukemia cells. Cancer Res. 1985,
45, 2050-2057.
(25) Bergeron, R. J .; McManis, J . S.; Weimar, W. R.; Schreier, K. M.;
Gao, F.; Wu, Q.; Ortiz-Ocasio, J .; Luchetta, G. R.; Porter, C.;
Vinson, J . R. T. The role of charge in polyamine analogue
recognition. J . Med. Chem. 1995, 38, 2278-2285.
(26) Porter, C.; Miller, J .; Bergeron, R. J . Aliphatic chain-length
specificity of the polyamine transport system in ascites L1210
leukemia cells. Cancer Res. 1984, 44, 126-128.
(27) Xia, C. Q.; Yang, J . J .; Ren, S.; Lien, E. J . QSAR analysis of
polyamine transport inhibitors in L1210 cells. J . Drug Targ.
1998, 6, 65-77.
Refer en ces
(1) Phanstiel, O., IV; Price, H. L,; Wang, L.; J uusola, J .; Kline, M.;
Shah, S. M. The Effect of Polyamine Homologation on the
Transport and Cytotoxicity Properties of Polyamine-(DNA-
Intercalator) Conjugates. J . Org. Chem. 2000, 65, 5590-5599.
(2) Wang, L.; Price, H. L.; J uusola, J .; Kline, M.; Phanstiel, IV, O.
The Influence of Polyamine Architecture on the Transport and
Topoisomerase II Inhibitory Properties of Polyamine DNA-
Intercalator Conjugates. J . Med. Chem. 2001, 44, 3682-3691.
(3) Wang, C.; Delcros, J .-G.; Biggerstaff, J .; Phanstiel IV, O. Syn-
thesis and Biological Evaluation of N1-(anthracen-9-ylmethyl)-
triamines as Molecular Recognition Elements for the Polyamine
Transporter. J . Med. Chem. 2003, 46, 2663-2671.
(4) Wang, C.; Delcros, J .-G.; Biggerstaff, J .; Phanstiel IV, O.
Molecular Requirements for Targeting the Polyamine Transport
System: Synthesis and Biological Evaluation of Polyamine-
Anthracene Conjugates. J . Med. Chem. 2003, 46, 2672-2682.
(5) Bergeron, R. J .; Feng, Y.; Weimar, W. R.; McManis, J . S.;
Dimova, H.; Porter, Carl; Raisler, B.; Phanstiel, O. A Comparison
of Structure-Activity Relationships between Spermidine and
Spermine Analogue Antineoplastics. J . Med. Chem. 1997, 40,
1475-1494.
(6) (a) Cullis, P. M.; Green, R. E.; Merson-Davies, L.; Travis, N.
Probing the mechanism of transport and compartmentalisation
of polyamines in mammalian cells. Chem. Biol. 1999, 6, 717-
729 and refs therein. (b) Seiler, N.; Dezeure, F. Polyamine
transport in mammalian cells. Int. J . Biochem. 1990, 22, 211-
218. (c) Seiler, N.; Delcros J -G.; Moulinoux, J . P. Polyamine
Transport in Mammalian Cells. An Update. Int. J . Biochem. Cell.
Biol. 1996, 28, 843-861.
(7) Kramer, D. L.; Miller, J . T.; Bergeron, R. J .; Khomutov, R.;
Khomutov, A.; Porter, C. W. Regulation of polyamine transport
by polyamines and polyamine analogues. J . Cell. Physiol. 1993,
155, 399-407.
(8) Sugiyama, S.; Matsuo, Y.; Maenaka, K.; Vassylyev, D. G.;
Matsushima, M.; Kashiwagi, K.; Igarashi, K.; Morikawa, K. The
1.8-A X-ray structure of the Escherichia coli PotD protein
complexed with spermidine and the mechanism of polyamine
binding. Protein Sci. 1996, 5, 1984-1990.
(9) Vassylyev, D. G.; Tomitori, H.; Kashiwagi, K.; Morikawa, K.;
Igarashi, K. Crystal structure and mutational analysis of the
Escherichia coli putrescine receptor. Structural basis for sub-
strate specificity. J . Biol. Chem. 1998, 273, 17604-17609.
(10) Kashiwagi, K.; Endo, H.; Kobayashi, H.; Takio, K.; Igarashi, K.
Spermidine-preferential uptake system in Escherichia coli. ATP
hydrolysis by PotA protein and its association with membrane.
J . Biol. Chem. 1995, 270, 25377-25382.
(11) Kashiwagi, K.; Miyamoto, S.; Nukui, E.; Kobayashi, H.; Igarashi,
K. Functions of potA and potD proteins in spermidine-prefer-
ential uptake system in Escherichia coli. J . Biol. Chem. 1993,
268, 19358-19363.
(12) Tomitori, H.; Kashiwagi, K.; Sakata, K.; Kakinuma, Y.; Igarashi,
K. Identification of a gene for a polyamine transport protein in
yeast. J . Biol. Chem. 1999, 274, 3265-3267.
(28) O′Sullivan, M. C.; Golding, B. T.; Smith, L. L. and Wyatt, I.
Molecular features necessary for the uptake of diamines and
related compounds by the polyamine receptor of rat lung slices.
Biochem. Pharm. 1991, 41, 1839-1848.
(29) Bergeron, R. J .; McManis, J . S.; Liu, C. Z.; Feng, Y.; Weimar,
W. R.; Luchetta, G. R.; Wu, Q.; Ortiz-Ocasio, J .; Vinson, J . R.
T.; Kramer, D.; Porter, C. “Antiproliferative Properties of
Polyamine Analogues: A Structure-Activity Study” J . Med.
Chem. 1994, 37, 4-3476.
(30) Soulet, D.; Covassin, L.; Kaouass, M.; Charest-Gaudreault, R.;
Audette, M.; Poulin, R. Role of endocytosis in the internalisation
of spermidine-C2-BODIPY,
a highly fluorescent probe of
polyamine transport. Biochemical J . 2002, 367, 347-357.
(31) Burns, M. R.; Carlson, C. L.; Vanderwerf, S. M.; Ziemer, J . R.;
Weeks, R. S.; Cai, F.; Webb, H. K.; Graminski, G. F. Amino Acid/
Spermine Conjugates: Polyamine Amides as Potent Spermidine
Uptake Inhibitors. J . Med. Chem. 2001, 44, 3632-3644.
(32) Mandel, J . L.; Flintoff, W. F. Isolation of mutant mammalian
cells altered in polyamine transport. J . Cell. Physiol. 1978, 97,
335-344.
(33) Byers, T. L.; Wechter, R.; Nuttall, M. E.; Pegg, A. E. Expression
of a human gene for polyamine transport in chinese hamster
ovary cells. Biochem. J . 1989, 263, 745-752.

5138 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Wang et al.
(34) (a) Chaney, S. E.; Kobayashi, K,.; Goto, R.; Digenis, G. A. Tumor
selective enhancement of radioactivity uptake in mice treated
with R-difluoromethylornithine prior to administration of 14C-
putrescine. Life Sci. 1983, 32, 1237-1241.(b) Heston, W. D. W.;
Kadmon, D.; Covey, D. F.; Fair, W. R. Differential effect of
R-difluoromethylornithine on the in vivo uptake of 14C-labeled
polyamines and methylglyoxal bis(guanylhydrazone) by a rat
prostate-derived tumor. Cancer Res. 1984, 44, 1034-1040.(c)
Redgate, E. S.; Grudziak, A. G.; Deutsch, M.; Boggs, S. S.
Difluoromethylornithine enhanced uptake of tritiated putrescine
in 9L rat brain tumors. Int. J . Radiat. Oncol. Biol. Phys. 1997,
38, 169-174.
(35) Porter, C. W.; Ganis, B.; Vinson, T.; Marton, L. J .; Kramer, D.
L.; Bergeron, R. J . Comparison and characterization of growth
inhibition in L1210 cells by alpha-difluoromethylornithine, an
inhibitor of ornithine decarboxylase, and N1, N8-bis(ethyl)-
spermidine, an apparent regulator of the enzyme. Cancer Res.
1986, 46, 6279-6285.
(36) (a) Kumar, C. V.; Asuncion, E. H. DNA Binding Studies and Site
Selective Fluorescence Sensitization of an Anthryl Probe. J . Am.
Chem. Soc. 1993, 115, 8547-8553. (b) Rodger, A.; Taylor, S.;
Adlam, G.; Blagbrough, I. S.; Haworth, I. S. Multiple DNA
Binding Modes of Anthracene-9-Carbonyl-N1-Spermine. Bioorg.
Med. Chem. 1995, 3, 861-872.
(37) Wang, C.; Abboud, K. A.; Phanstiel, O. IV Synthesis and
Characterization of N1-(4-toluenesulfonyl)-N1-(9-anthracene-
methyl)triamines. J . Org. Chem. 2002, 67, 7865-7868.
(38) Blagbrough, I. S.; Geall, A. J . Practical Synthesis of Unsym-
metrical Polyamine Amides. Tetrahedron Lett. 1998, 439-442.
(39) Kuksa, V.; Buchan, R.; Lin, P. K. T. Synthesis of Polyamines,
Their Derivatives, Analogues and Conjugates. Synthesis 2000,
No. 9, 1189-1207.
Posse´me´, F.; Le Barbier, C.; Carreaux, F.; Carboni, B.; Seiler,
N.; Moulinoux, J .-P.; Delcros, J .-G. N-Benzylpolyamines as
Vectors of Boron and Fluorine for Cancer Therapy and Imag-
ing: Synthesis and Biological Evaluation. J . Med. Chem. 2001,
44, 3653-3664.
(41) Cheng, Y.-C.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes
50% inhibition (IC50) of an enzymatic reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
(42) Bergeron, R. J .; Mu¨ller, R.; Bussenius, J .; McManis, J . S.;
Merriman, R. L.; Smith, R. E.; Yao, H.; Weimar, W. R. Synthesis
and Evaluation of Hydroxylated Polyamine Analogues as Anti-
proliferatives. J . Med. Chem. 2000, 43, 224-235.
(43) Covassin. L.; Desjardins, M.; Charest-Gaudreault, R.; Audette,
M.; Bonneau, M. J .; Poulin, R. Synthesis of spermidine and
norspermidine dimers as high affinity polyamine transport
inhibitors. Bioorg. Med. Chem. Lett. 1999, 9, 1709-1714.
(44) (a) Fransson, L.-A. Glypicans. Int. J . Biochem. Cell Biol. 2002,
1348, 1-5. (b) Belting, M.; Borsig, L.; Fuster, M. M.; Brown, J .
R.; Persson, L.; Fransson, L.-A.; Esko, J . D. Tumor attenuation
by combined heparan sulfate and polyamine depletion. Proc.
Natl. Acad. Sci. U.S.A. 2002, 99, 371-376. (c) Belting, M.;
Persson, S.; Fransson, L.-A. Proteoglycan involvement in
polyamine uptake. Biochem. J . 1999, 338, 317-323.
(45) Mislick, K. A.; Baldeschwieler, J . D. Evidence for the role of
proteoglycans in cation-mediated gene transfer. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 12349-12354.
(46) Mounkes, L. C.; Zhong, W.; Cipres-Palacin, G.; Heath, T. D.;
Debs, R. J . Proteoglycans Mediate Cationic Liposome-DNA
Complex-based Gene Delivery in Vitro and in Vivo. J . Biol.
Chem. 1998, 273, 26164-26170.
(47) Mosmann, T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J .
Immunol. Methods 1983, 65, 55-63.
(48) Cle´ment, S.; Delcros, J . G.; Feuerstein, B. G. Spermine uptake
is necessary to induce haemoglobin synthesis in murine erythro-
leukemia cells. Biochem. J . 1995, 312, 933-938.
(49) Torossian, K.; Audette, M.; Poulin, R. Substrate protection
against inactivation of the mammalian polyamine transport
system by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Bio-
chem. J . 1996, 319, 21-26.
(40) (a) Pegg, A. E. The role of polyamine depletion and accumulation
of decarboxylated S-adenosylmethionine in the inhibition of
growth of SV-3T3 cells treated with alpha-difluoromethyl-
ornithine. Biochem. J . 1984, 224, No. 1, 29-38. (b) Seiler, N.;
Kno¨dgen, B. Determination of polyamines and related com-
pounds by reversed-phase high-performance liquid chromatog-
raphy: Improved separation systems. J . Chromatography 1985,
339, 45-57. (c) Gaboriau, F.; Havouis, R.; Moulinoux, J .-P.;
Delcros, J .-G. Atmospheric pressure chemical ionization-mass
spectrometry method to improve the determination of dansylated
polyamines. Anal. Biochem. 2003, 318, 212-220. (d) Martin, B.;
J M030223A