Molecular requirements for targeting the polyamine transport system. Synthesis and biological evaluation of polyamine-anthracene conjugates

Article abstract of DOI:10.1021/jm020598g

A series of nine N¹-(9-anthracenylmethyl)tetraamines (e.g., Ant-4,4,4-tetraamine) were synthesized and evaluated for cytotoxicity in L1210, α-difluoromethylornithine (DFMO)-treated L1210, Chinese hamster ovary (CHO), and CHO-MG cell lines. Surp

Full text of DOI:10.1021/jm020598g

2672
J . Med. Chem. 2003, 46, 2672-2682
Molecu la r Requ ir em en ts for Ta r getin g th e P olya m in e Tr a n sp or t System .
Syn th esis a n d Biologica l Eva lu a tion of P olya m in e-An th r a cen e Con ju ga tes
Chaojie Wang,†,‡ J ean-Guy Delcros,§ J ohn Biggerstaff,† and Otto Phanstiel, IV*,†
Groupe de Recherche en Therapeutique Anticance´reuse, Faculte´ de Me´decine, 2, Avenue du Professeur Le´on Bernard,
35043 Rennes, France, and Department of Chemistry, P.O. Box 162366, University of Central Florida,
Orlando, Florida 32816-2366
Received J anuary 2, 2003
A series of nine N1-(9-anthracenylmethyl)tetraamines (e.g., Ant-4,4,4-tetraamine) were syn-
thesized and evaluated for cytotoxicity in L1210, R-difluoromethylornithine (DFMO)-treated
L1210, Chinese hamster ovary (CHO), and CHO-MG cell lines. Surprisingly, the 3,3,4- and
3,4,3-tetraamine motifs had the same or decreased cytotoxicity in DFMO-treated L1210 cells,
whereas the rest of the tetraamine systems were usually more cytotoxic and gave lower IC50
values in this treated cell line. The most sensitive derivatives to DFMO treatment were the
Ant-4,4,3- and Ant-4,4,4-tetraamine analogues, which were 7 and 5 times more cytotoxic in
DFMO-treated L1210 cells, respectively. Ki values for each of the anthracenylmethyl(Ant)-
polyamine conjugates were determined in L1210 cells and revealed that these systems are
high-affinity ligands for the polyamine transporter (PAT). Mixed results were observed in the
CHO and CHO-MG assays. The 4,4,4- and 5,4,4-tetraamine motifs were 3 times more toxic to
CHO cells with active polyamine transporters. For example, the Ant-4,4,4-tetraamine conjugate
displayed IC50 values of 11 µM in CHO cells and 33 µM in CHO-MG cells, a PAT-deficient cell
line. This suggested that these derivatives used the PAT in part to access cells. However, most
of the other tetraamine derivatives had similar potencies in both the CHO and CHO-MG cell
lines. In terms of vector design, higher affinity for the PAT (lower Ki values) did not translate
into higher potency for the tetraamine conjugate. In contrast, the related triamine systems,
which had micromolar Ki values in L1210 cells, were more efficacious and selective. In one
case, the 4,4-triamine motif imparted 150-fold higher potency in CHO cells than the CHO-MG
mutant. A deconvolution microscopy study in A375 melanoma cells revealed a rapid internaliza-
tion of the Ant-4,4-triamine as fluorescent vesicles, whereas the Ant-4,4,4-tetraamine remained
mostly at the cell surface. These findings help define the key characteristics required for
selective delivery of polyamine-drug conjugates into cell types with active polyamine
transporters.
In tr od u ction
augmented by polyamine chain extension. This report
investigated the related N1-substituted tetraamine sys-
tems (e.g., 1, where R ) -(CH2)oNH2) and identified
important biophysical parameters required to use con-
jugates between toxic agents (anthracene) and the
native polyamines (e.g., putrescine, spermidine, and
spermine; 2-4 in Figure 1) as vector systems.
While the terminally bis-alkylated polyamines have
yielded diverse biological activity ranging from anti-
cancer to antidiarrheal agents,5,11 their monosubstituted
analogues have limited publications describing their use
in vector design.1,2,5,12,13,16-23 This, in part, may stem
from their less direct syntheses, which involve several
steps.5,24,25 The conjugates in this report comprise an
anthracene nucleus covalently bound to a tetraamine
framework. In this manner, cells will be challenged to
import the same lethal cargo (anthracene) using various
polyamine motifs to assist in cellular entry.1,2,13
The anthracene component was selected because of
significant preliminary data, which revealed its in-
creased cytotoxicity (lower IC50 value) over an acridine
analogue in murine leukemia (L1210) cells.1,2,12,13 The
IC50 value is the concentration of the drug required to
kill 50% of the cell population. Moreover, the anthracene
provides a convenient UV “probe” for compound iden-
tification and elicits a toxic response from cells upon
Vectored or targeted drugs, which have enhanced
affinity for cancer cells, would be an important advance
in cancer chemotherapy. The polyamine transport sys-
tem offers one method of targeting specific cell types
via the molecular recognition events involved during the
importation of exogenous polyamines.1-13 Prior work in
our laboratories evaluated the cytotoxicity and polyamine
transporter (PAT) affinity of conjugates containing
branched polyamine1,2 and linear triamine motifs at-
tached to either an anthracene or acridine nucleus.12,13
Indeed, the preceding article identified certain linear
triamines as excellent vectors.13 In particular, the 4,4-
triamine system 1 (Figure 1; 1, where R ) H and both
m and n are 2) had 150-fold higher cytotoxicity in
Chinese hamster ovary (CHO) cells (which contained
an active polyamine transport system) than in a mutant
CHO-MG cell line, which was PAT-deficient.14,15 We
wondered whether this exquisite selectivity for cell types
with active polyamine transporters could be further
* To whom correspondence should be addressed. E-mail: ophansti@
mail.ucf.edu. Phone: (407) 823-5410. Fax: (407) 823-2252.
† University of Central Florida.
‡ C.W. is a visiting scholar from the Department of Chemistry,
Henan University, Kaifeng, 475001, P. R. China.
§ Groupe de Recherche en Therapeutique Anticance´reuse, Faculte´
de Me´decine.
10.1021/jm020598g CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/20/2003

Polyamine-Anthracene Conjugates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2673
F igu r e 1. Sample conjugate system, 1, the native polyamines (2-4), and DFMO, 5.
Sch em e 1
entry (presumably through DNA coordination).13,26,27
Therefore, a series of tetraamine-anthracene conju-
gates were synthesized and screened for uptake via the
PAT. By judicious choice of amine substrates (e.g., a
synthetic library) and proper biological experiments
(e.g., IC50 and Ki measurements) for selected cell types
(murine leukemia L1210, CHO, and CHO-MG cells), one
can now comment on transporter affinity and cytotox-
icity as a function of polyamine-drug conjugate struc-
ture.
varying chain lengths between the nitrogen centers.
Prior work involved the use of amino alcohols as
precursors to polyamines.13,28 Sequential reactions of
O-tosylation, tosylate displacement by an amino alcohol
(or a diamine), and tert-butyloxycarbonylation (Boc
introduction) allowed for the rapid construction of the
desired scaffolds.13
The tosylates 6 were best generated and used as soon
as possible.13 The tosylated compounds 6a -c were
reacted with excess amino alcohols to form several
derivatives, 7a -f, which were purified and character-
ized. The triamines 1a -f were synthesized from 6 as
described in the preceding article.13 The target com-
pounds 11a -i were prepared by an analogous route as
shown in Scheme 1.
Resu lts a n d Discu ssion
Syn th esis. As shown in Scheme 1, the synthesis of
the tetraamine conjugates involved several steps. The
modular approach allowed for the introduction of

2674 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Wang et al.
F igu r e 2. Polyamine biosynthetic pathway.
The tandem Boc introduction and tosylation sequence
followed by diamine monoalkylation afforded 10 in good
yields. All the N-Boc protected compounds 8 (except 8c)
were isolated and characterized. To streamline this
multistep sequence, the crude product mixtures were
used in the reaction scheme. For example, the crude
product of 8c was used directly in the synthesis of 9c.
The impurities present in 8c did not affect the subse-
quest tosylation step. Therefore, one could avoid the
tedious column purification of 8. In fact, it was not
necessary to isolate the tosylates 9 either. Nevertheless,
two (9b, 9d ) of the six compounds of type 9 were isolated
to confirm their structure. Using the crude products of
9 for the next substitution step worked well, as expected
from our prior success with the alkylation of amino
alcohols.13 The partial N-Boc protected tetraamines 10
were prepared in satisfactory yields. They were con-
sumed immediately after purification to get the nine
target tetraamines 11 as their HCl salts, which were
purified conveniently by washing the solids with abso-
lute ethanol.
Ta ble 1. Comparison of Unsubstituted Polyamine Ki Values of
L1210 Cells
Ki values (nM)
compd (tether)
of L1210 cells
1,4-diaminobutane 12 (putrescine)
1,8-diaminoctane 13
(3,5) triamine 14
(3,3) triamine 15
(3,4) triamine 16 (spermidine)
(4,4) triamine 17
(3,3,3) tetraamine 18
(3,4,3) tetraamine 19 (spermine)
(4,4,4) tetraamine 20
208200 ( 16340
24090 ( 780
8030 ( 500
5120 ( 210
2460 ( 507a
2300 ( 80
1510 ( 120
1340 ( 310
730 ( 60
a
Km value.
An efficient method for the multistep synthesis of the
N-monosubstituted tetraamines was developed. The Boc
protection and tosylation steps each proceeded in high
yield, and the crude products of these compounds could
be used directly in the next step. This avoided the lower
yields due to isolation steps (with 8 and 9) and provided
for a more efficient synthesis. The total yields of the
three steps from intermediates 7 to 10 were generally
between 65% and 85% and avoided column chromatog-
raphy. In short, the tetraamines 11a -i were generated
in approximately 30% overall yield from the starting
aldehyde.
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
CHO cells were chosen along with a mutant cell line
(CHO-MG) in order to comment on selective transport
via the PAT.12
R-Difluoromethylornithine (DFMO, 5 in Figure 1) is
a known ornithine decarboxylase (ODC) inhibitor.30
ODC is the enzyme responsible for the biosynthesis of
putrescine from L-ornithine (see Figure 2). Putrescine
(PUT, 2) is then converted to the longer polyamines,
spermidine (SPD, 3) and spermine (SPM, 4), via suc-
cessive aminopropylation steps (Figure 2). Inhibition of
ODC blocks intracellular putrescine biosynthesis and
leads to a significant increase in polyamine uptake,
which allows the cell to acquire polyamines from exog-
enous sources. Therefore, cells, which are treated with
DFMO, should be more susceptible to the polyamine-
vectored conjugates and should provide lower IC50
values.11-13 IC50 values in L1210 cells with and without
DFMO treatment were determined. Conjugates that
selectively target the PAT should be more potent with
DFMO-treated cells and should provide L1210/(L1210
+ DFMO) IC50 ratios greater than 1.
The Ki value is a measure of the affinity of the
polyamine conjugate for the polyamine transporter and
is determined in a competitive assay with radiolabeled
spermidine. 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 relation-
ship with our synthetic ligands. At present, we were
unable to discreetly measure each of these modes of
import. Therefore, our Ki measurements reveal the
overall transporter affinity of each synthetic conjugate
when all these import modes are active in L1210 cells.
These collective polyamine import modes will be re-
ferred to as the “polyamine transporter” or PAT.
As shown in Table 1, the innate affinities of unsub-
stituted polyamine systems (12-20) for the PAT (Ki
values) were measured. The following trend toward
increasing transporter affinity (lower Ki value) was
observed: diamines < triamines < tetraamines. In
addition, the tether (number of CH2 spacer units)
between the nitrogen centers had a dramatic effect on
the Ki value (e.g., 12 vs 13). A clear preference for the
aminobutyl spacer was evident (e.g., 15 vs 17 and 18
vs 20). With this preliminary information, we expected
a range of activity for the synthetic conjugates 1 and
11. Indeed, diverse biochemical parameters were ob-
tained with the fluorescent PAT probes listed in Table
2.
The IC50 values listed in Table 2 revealed that L1210
cells were more susceptible to triamine conjugates 1a -f
than the corresponding tetraamines 11. This was sur-
prising because the Ki values for the tetraamines
(nanomolar range) were significantly lower than the

Polyamine-Anthracene Conjugates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2675
Ta ble 2. Biological Evaluation of Triamines 1a -f13, Tetraamines 11a -i, Other Published Analogues 21-25,31,12 and Control 2813 in
L1210 Cellsa
L1210
IC50 (µM)
L1210 + DFMO
IC50 (µM)
L1210/(L1210 + DFMO)
L1210
Ki (nM)
compd (tether)
1a (3,3)
1b (3,4)
1c (4,3)
1d (4,4)
1e (5,3)
1f (5,4)
11a (3,3,4)
11b (3,4,3)
11c (3,4,4)
11d (3,5,4)
11e (4,4,3)
11f (4,4,4)
11g (5,4,3)
11h (5,4,4)
11i (5,5,4)
IC50 ratio
1.8 ( 0.4
0.7 ( 0.3
0.4 ( 0.1
0.3 ( 0.04
1.3 ( 0.1
0.4 ( 0.1
21.8 ( 3.2
19.5 ( 2.8
10.2 ( 1.6
10.7 ( 2.4
4.3 ( 0.6
7.5 ( 0.3
6.4 ( 1.0
7.2 ( 0.6
7.1 ( 1.2
>100
1.4 ( 0.3
0.3 ( 0.1
0.2 ( 0.02
0.15 ( 0.1
0.7 ( 0.1
0.3 ( 0.1
21.9 ( 4.3
31.9 ( 1.9
5.1 ( 0.6
7.2 ( 0.1
0.6 ( 0.2
1.6 ( 0.3
2.1 ( 0.7
3.8 ( 0.1
4.3 ( 0.9
ND
1.3
2.3
2
33350 ( 2630
2500 ( 260
6230 ( 580
1750 ( 140
5020 ( 580
1650 ( 230
108 ( 13
202 ( 8
80 ( 9
2
1.9
1.3
1.0
0.6
2.0
1.5
7.2
4.7
3.1
1.9
1.6
ND
ND
ND
8.1
3.8
0.7
90 ( 6
74 ( 5
51 ( 6
99 ( 8
65 ( 5
64 ( 5
21 N-ethyl (3,3,3)
22 N-ethyl (3,4,3)
23 N-ethyl (4,4,4)
24 (3,4,3) amidoacridiny
25 (3,4,3) aminoacridinyl
28 (N-butyl) control
7700
99
ND
ND
2.2
0.6
1700
1100
83
83
14
18
2.3
14.6 ( 0.1
21.9 ( 3.6
62300 ( 4200
a
IC50 values were determined after 48 h of incubation with the conjugate. The Ki value of SPM is 1.34 µM, and the Km value of SPD
is 2.46 µM.12 ND ) not determined.
values for the triamine systems (micromolar range). The
lower Ki values in Table 2 reflect the higher affinity of
the polyamine derivative for the polyamine transporter
on the cell surface. The trend clearly indicated a much
higher affinity of the tetraamines for the PAT. A priori,
one may have anticipated a direct correlation between
low Ki values and low IC50 values (i.e., high affinity PAT
ligands should give more potent drugs). Ironically, the
opposite trend was observed!
The presence of DFMO resulted in increased potency
(lower IC50 values in Table 2) for all conjugates 1 and
11 except 11a and 11b. Dramatic increases in potency
were observed for specific tetraamines, 11e-g. The most
sensitive substrates 11e and 11f were 7 and 5 times
more cytotoxic in the presence of DFMO, respectively.
The results with the other tetraamine systems 11a -d
and 11g-i were mixed. With the exception of 1a and
1f, the related triamine systems were usually twice as
potent in L1210 cells with DFMO treatment.13 These
results suggest that both triamine and tetraamine
analogues (1 and 11) can use the PAT to access cells.
Why are the tetraamine analogues not more potent?
An elementary answer to this dilemma is that the
anthracene-triamine architecture is simply more toxic
to the cell than the related anthracene-tetraamine
construct. This interpretation would assume that the
polyamine architecture itself is toxic to the cell and that
different polyamine scaffolds will have different cyto-
toxicities. However, a more intriguing answer may lie
in how efficiently these materials (e.g., 1 or 11) 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 in Table 1, the latter premise
seems to be the most reasonable. In other words, the
polyamine “message” is paramount to PAT recognition.
Several scenarios may be considered. Even though the
N-substituted tetraamines (e.g., 11) have high affinity
for the PAT, inefficient import (very low Vmax) could
explain their reduced cytoxicity. Alternatively, N-sub-
stituted tetraamines may inhibit their own transport
(as has been reported for other tetraamine deriva-
tives).12
An assessment of the conjugates’ ability to target the
PAT was conducted in two CHO cell lines. The CHO-
MG cell line is polyamine-transport-deficient and was
isolated for growth resistance to methylglyoxalbis-
(guanylhydrazone) using a single-step selection after
mutagenesis with ethylmethane sulfonate.14 For the
purposes of this study, the CHO-MG cell line repre-
sented cells with limited polyamine transporter activity
and provides a measure of delivery independent of the
PAT. In contrast, the parent CHO cell line represents
cell types with active polyamine transport.14,15 Com-
parison of conjugate potency 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.12,14 Therefore, IC50 determina-
tions in these two CHO lines provided a relative ranking
of delivery via the PAT. In short, highly selective,
vectored conjugates should give high CHO-MG/CHO
IC50 ratios.
As shown in Table 3, the triamines were usually more
potent than the tetraamines in CHO cells. Biological
evaluation of triamines 1a -f and tetraamines 11a -i
in the two CHO cell lines also revealed striking prefer-
ences by certain polyamine architectures for the PAT.
As mentioned previously, the 4,4-triamine 1d displayed
nearly 150-fold higher cytotoxicity in the CHO over the
CHO-MG cell line.13 In contrast, the most selective
tetraamines 11f and 11h were only 3-fold more potent
in the CHO cell line. These findings demonstrate that
CHO and CHO-MG IC50 comparisons are an excellent
way to rank polyamine vectors and their transport.12
The cytotoxicity of the triamines 1 in the CHO lines
correlated nicely with the L1210 IC50 and Ki results.13
Therefore, the triamine systems seem to be well-
behaved systems that give consistent data in all three
cell lines.

2676 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Wang et al.
Ta ble 3. Biological Evaluation of Triamines 1a -f,13
Tetraamines 11a -i, Other Published Conjugates 24 and 25,12
and Control 2813 in CHO-MG and CHO Cell Lines
Comparison of 11b (Ant-3,4,3) with 24 and 25 sug-
gests that the distally tethered acridine motifs present
in 24 and 25 impart higher potency than the anthra-
cenylmethyl substituent when tested in CHO cells
(Table 3). Interestingly, DFMO treatment of L1210 cells
resulted in the expected increase in potency of 24 and
25 but surprisingly gave decreased cytotoxicity for 11b
(Table 2). This seems to be an anomaly and unique for
the 3,4,3-tetraamine motif in 11b, since it was the only
entry in Table 2 with an IC50 ratio of <1. Ironically, this
3,4,3 motif is equivalent to N1-substituted spermine
(SPM), a native polyamine. The other polyamine homo-
logues all had IC50 ratios of g1 in Table 2. The
differences in cytotoxicity (between 11b, 24, and 25)
may stem from the different degrees of conformational
freedom imparted by the chain length available to the
longer tether of the acridine substitutent as well other
modes of cellular entry for the acridine ring system.1,2,12
A more direct structural comparison of the anthrace-
nylmethyl and acridine nuclei was conducted previously
with the branched polyamine motifs 26 and 27.1,2
Earlier work demonstrated that the N-(9-anthracenem-
ethyl) substituent of 26 was more potent than the
corresponding N-(9-aminoacridinyl) substituent in 27
(47 h IC50 values of 13 µM for 26 and 75 µM for 27).1,2
Nevertheless, the fact that 24 and 25 were more
cytotoxic than 11b suggested that further structural
modification is accommodated by the PAT as long as
the proper “polyamine message” is in place.
CHO-MG
CHO
CHO-MG/CHO
IC50 ratio
compd (tether)
1a (3,3)
1b (3,4)
1c (4,3)
1d (4,4)
1e (5,3)
1f (5,4)
11a (3,3,4)
11b (3,4,3)
11c (3,4,4)
11d (3,5,4)
11e (4,4,3)
11f (4,4,4)
11g (5,4,3)
11h (5,4,4)
11i (5,5,4)
IC50 (µM)
IC50 (µM)
3.4 ( 0.5
8.8 ( 1.2
9.5 ( 1.1
1.9 ( 0.4
2.5 ( 0.7
0.4 ( 0.1
1.8
3.5
24
148
2.5
38
0.9
1.3
1.7
1.2
0.7
3.1
1.9
3.1
1.4
1.2
3.1
1.1
66.7 ( 4.1 0.45 ( 0.1
10.1 ( 1.2 4.1 ( 0.5
57.3 ( 2.9 1.5 ( 0.1
41.5 ( 3.5 44 ( 0.0
75.7 ( 7.4 59.7 ( 6.5
52.8 ( 2.6 31.1 ( 7.3
41.7 ( 0.2 34.9 ( 1.3
2.8 ( 0.4
4.0 ( 1.4
33.2 ( 1.7 10.6 ( 0.0
33.5 ( 3.5 18.1 ( 3.5
30.8 ( 0.4 9.9 ( 1.6
5.7 ( 1.6
4.0 ( 0.8
24 (3,4,3) amidoacridinyl 21
25 (3,4,3) aminoacridinyl 7.2
17
2.3
28 (N-butyl) control
11.2 ( 2.3 10.5 ( 2.0
In contrast, the tetraamines were more complex and
few guiding trends were evident. For example, the 4,4,4
tetraamine 11f had the highest affinity for the PAT as
evidenced by its low Ki value (51 nM) in L1210 cells,
but this feature only correlated with a 3-fold increase
in cytotoxicity with CHO cells over their CHO-MG
counterparts. In short, the high PAT affinity of these
tetraamine ligands did not translate to dramatic in-
creases in cytotoxicity in either the L1210 or CHO cell
lines.
It should be noted that the control N-butyl derivative
2813 (shown in Figure 3) gave no enhancement in
cytotoxicity when dosed in the presence of DFMO and
had a high Ki value (62 300 nM, Table 2) in L1210 cells.
Thus, an N-butylanthracene moiety like 28 has little
affinity for the PAT and likely enters the cell via another
pathway. Moreover, as shown in Table 3, this N-butyl
control showed virtually no preference in targeting CHO
over CHO-MG cells. Collectively, these observations
further support the ability of certain cell types to
recognize the conjugates via their polyamine motifs.
In terms of vector design, the 4,4-triamine motif
(homospermidine) was found to be the optimal chemical
signal to gain entry to cells via the PAT. For example,
conjugates 1d , 11c, 11f, and 11h each present the
terminal 4,4-triamine “signal” to the PAT receptor and
each gave the highest selectivity, i.e., the highest (CHO-
MG/CHO) IC50 ratio, in their respective classes. Of these
four, the triamine 1d clearly gave the best selectivity
profile. Furthermore, 1d had relatively low toxicity in
cells with low PAT activity and high toxicity in cells with
high polyamine transport activity.
Are tight binders such as the tetraamines 11 localized
at the cell membrane and internalized more slowly? A
possible answer is that the tetraamines, in general,
rapidly suppress PAT activity and limit their own
uptake by the transporter.12 This premise is supported
by the observed low Ki values (both were 83 nM) and
low CHO-MG/CHO IC50 ratios for 24 (1.2) and 25 (3.1),
which are similar to those of the other tetraamines
tested (11).
Other authors have shown differences in transporter
affinity (Ki) and L1210 cytotoxicity (IC50) behavior for
related N-monoalkyltetraamine architectures.31 As shown
in Table 2, the terminal monoethylated tetraamines 21,
22, 23 were found by Bergeron et al. to have Ki values
in the micromolar range (7.7, 1.7, and 1.1 µM, respec-
tively) and higher 48 h IC50 values (g14 µM). The
N-ethyl substrates 22 and 23 allow for direct compari-
son to our findings with the N-anthracenylmethyl 3,4,3-
tetraamine 11b and the 4,4,4-tetraamine 11f. A priori
one may have expected that polyamines with bulky N
substituents would be poor ligands for the polyamine
transporter. However, the comparison suggests that the
bulky anthracenylmethyl substituent actually enhances
the PAT binding affinity and cytotoxicity of the polyamine
conjugate in vitro. As shown in Table 2, the tetraamines
11, 24, and 25 have nanomolar Ki values in L1210 cells.
Delcros and co-workers have shown a similar effect with
N1-acridinyl substituted tetraamines.12 Therefore, N1-
substitution with an appended aromatic ring system like
the anthracenylmethyl or acridinyl unit leads to a large
decrease in the Ki value (see Table 2 for Ki values for
22 vs 11b, 24, and 25). In short, aromatic substituents
seem to impart higher affinity for cell-surface receptors.
To further address this issue, we conducted a qualita-
tive study using deconvolution microscopy to compare
the relative rates of delivery of the 4,4-triamine 1d and
the 4,4,4-tetraamine 11f “probes” into A375 melanoma
cells. Melanoma cells were chosen because of their large
size and susceptibility to polyamine analogues.6 A375
cells were incubated with the respective conjugate (at
10 µM) for 3 min. A series of optical “slices” of the
resultant cells were obtained, and one slice was chosen
to best represent the sample overall. The anthracene
component was easily tracked via its diagnostic fluo-
rescence properties and is shown in white and gold in
Figure 4. The UV spectra of the anthracene conjugates
suggest a consistent λmax near 364 nm and an emission
maxima near 417 nm. The relative levels of fluorescence

Polyamine-Anthracene Conjugates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2677
F igu r e 4. Deconvolution microscopy images of A375 cells
treated with 1d (panels 1and 2) and 11f (panels 3 and 4).
Panels 1 and 3 represent cells incubated with 1d and 11f,
respectively, at 10 µM for 3 min and then fixed with paraform-
aldehyde and washed with buffer. Panels 2 and 4 represent
cells that were incubated with 1d and 11f, respectively,
washed with buffer, and then fixed with paraformaldehyde.
why the tetraamines are less potent. The high-affinity
tetraamine ligands bind rapidly to the cell surface by
interacting with the PAT and other cell-surface recep-
tors. Moreover, they appear to be less internalized than
the related triamines. Indeed, slower uptake is likely
related to their lower potency. The observation that the
high-affinity tetraamine ligands remain bound to the
cell surface even after washing is also noteworthy. This
finding provides a rationale for earlier observations
wherein PAT activity was not restored upon removal
of the tetraamine drug from the culture medium.12
Indeed, this “sticky tetraamine” phenomenon may be a
property of N-substituted tetraamines in general.
There is a strong possibility that the tetraamines 11
are associated with other non-PAT receptors on the cell
surface. As seen previously in panel 4, the widespread
distribution of 11f remaining across the cell surface
(even after washing) suggests that numerous nonlocal-
ized interactions occurred between 11f (at 10 µM) and
the cell membrane. This observation was not surprising
because polyamines have a high affinity for membrane
constituents (such as phospholipids and glycoproteins)32
that is strongly dependent on the number of amino
groups. Moreover, the large aromatic moiety should
favor bridging interactions between both proteins and
lipids in the membrane. Earlier work by Delcros and
others revealed that L1210 cells were still susceptible
to tetraamine-acridine conjugates (like 25) even in the
presence of 500 µM spermidine (SPD). On the basis of
the Ki values determined, this quantity of SPD should
have completely inhibited the uptake of the conjugate
25. Indeed, excess SPD in the culture medium only
partially reduced the amount of 25 associated with the
cells.12 This finding suggested that 500 µM SPD was
F igu r e 3. Other polyamine conjugates 21-27 and the N-butyl
control 28.
suggested different internalization rates and localization
behavior between the two probes (1d and 11f). Panels
1 and 3 in Figure 4 are control samples, wherein after
incubation with the respective polyamine conjugate, the
cells were first fixed with paraformaldehyde and then
washed. Copious amounts of triamine 1d (panel 1) or
11f (panel 3) remain associated with the cell surface.
However, when the cells were first washed with fresh
buffer and then fixed with paraformaldehyde, a different
result was obtained. As shown in Figure 4, the triamine
conjugate 1d (panel 2) clearly accessed the cell more
rapidly than 11f (panel 4) as evidenced by the greater
number of fluorescent vesicles inside the cell. The
triamine probe 1d was mainly sequestered as internal
vesicles, whereas the tetraamine counterpart 11f was
nearly exclusively bound to the cell surface with few
vesicles being formed during this short time period. The
limited amount of triamine at the surface is consistent
with the lower binding affinity of this probe to PAT
(Table 2) and presumably to other membrane receptors.
A comparison of panels 1 and 2 (with 1d ) revealed that
both the surface-bound and unbound triamines were
more efficiently washed away from the cell surface in
the workup step.
These findings are significant because they are the
first comparative observations of polyamine vectors in
action. Moreover, they offer a possible explanation of

2678 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Wang et al.
unable to outcompete for the bound N-substituted
tetraamine sites and that unsubstituted triamines such
as SPD have a lower affinity for these alternative
tetraamine docking sites. Moreover, prior work by other
authors has shown that spermine (SPM, i.e., the 3,4,3-
tetraamine) reduces the lateral mobility of glycoproteins
inside the membrane.33 In addition, Ballas has shown
that interactions between an exogenous tetraamine
(SPM) and erythrocyte membranes resulted in de-
creased membrane deformability.33 Because the N1-aryl-
substituted tetraamines likely have a much higher
affinity for the membrane than SPM, they should have
a greater influence on the membrane. Strong binding
of tetraamines may impair both the membrane struc-
ture and its functions. Such an event may ultimately
limit tetraamine import.
On the other hand, N1-substituted triamines such as
1d are bound less tightly to the cell surface. As shown
in Figure 4 (panels 1 and 2), the loosely bound triamines
can be washed off the cell surface with aqueous buffer.
The triamines likely act as reversible ligands, which are
more selective in bonding to their cellular target, the
PAT. In an anthropomorphic sense, the triamines
present their “polyamine message” or “passport” to
various cellular gates of entry. They reversibly search
along the cell surface and rapidly enter the correct gate.
In contrast, the tetraamines present “valid passports”
to numerous surface targets (gates) and are held tightly
to the cell surface as they interact with these numerous
“gates”. These multiple binding events likely disrupt the
stability of the membrane and result in slow tetraamine
entry into the cell. This speculation is supported some-
what by Figure 4 (panel 4), wherein the tetraamine
remains bound to a large portion of the cell surface (even
after a buffer wash) and fewer vesicles are apparent
inside the cell (compared to panel 2). Another explana-
tion may be that the tetraamine interacts strongly with
the PAT gate itself and limits its own import. Regardless
of the rationale, the lower PAT affinity of the triamines
provided a more selective vector.
a role in polyamine transport.32,35,36 Proteoglycans are
composed of glycosaminoglycan chains covalently linked
to protein, and expression of cell-surface proteoglycans
is required for the binding of many extracellular sub-
strates.32 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.32 The data indicated a
specific role for heparan sulfate proteoglycans (HSPG)
in the uptake of spermine by fibroblasts. These provoca-
tive results suggest that proteoglycan-mediated poly-
amine transport may play an important role in future
drug delivery strategies.37,38 The current report also
provides new fluorescent probes needed to evaluate
these interesting new pathways.
In summary, the polyamine transporter is capable of
distinguishing between isomeric systems that have the
same overall length but a different separation of point
charges.5,31,39-42 The current study has identified some
of the optimal motifs and the necessary biophysical
parameters in order to initiate transport via this
pathway.
Con clu sion s
An efficient modular synthesis of N1-substituted
polyamines containing different tether lengths between
nitrogen centers was developed. This new methodology
should contribute to the limited synthetic approaches
used to access linear polyamines.43 All triamine conju-
gates 1 and most tetraamines (except 11a and 11b) had
higher potency in DFMO-treated L1210 cells. High PAT
affinity (low nanomolar Ki values) did not translate into
a more efficacious drug. A survey of different polyamine
vectors revealed that the respective 4,4- and 5,4-
triamine systems had 150-fold and 38-fold selectivity
for CHO cells containing active polyamine transporters.
Optimal use of this transporter seems to require a
balance involving moderate PAT binding affinity and
conjugate internalization. Future studies will use these
fluorescent polyamine probes to further delineate the
delivery differences. In summary, selective vector motifs
were identified that ferry drugs into cancer cell types
via the PAT.
The findings are also consistent with a model pro-
posed by Cullis and Poulin.6,34 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
compartment via a cationic symporter.6 Indeed, panel
4 in Figure 4 seems to have captured this endocytosis
event in action. According to this model, in order to exit
the vesicle (via the putative cationic symporter), the
conjugate must dissociate from its vesicular PAT recep-
tor. Since the tetraamines are high-affinity PAT ligands,
they likely dissociate more slowly from the receptor. In
contrast, the triamines have lower binding affinities and
should dissociate more rapidly from the internalized
PAT receptor to exert their toxic effect. Although further
kinetic studies are needed, this rationale would also
explain why the triamines are more potent and yet have
higher Ki values.
Because many cancer cell lines have high PAT activ-
ity, especially when dosed with DFMO,6,44-46 these
results suggest a new anticancer strategy predicated
upon the use of a triamine vector to shuttle appended
drug “cargoes” selectively into cells. Future work will
evaluate this strategy in a mouse leukemia model.
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
percent, and NH4OH refers to concentrated aqueous NH4OH.
All tested compounds provided satisfactory elemental analyses,
which 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.
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. However, recent reports by Frannson
and co-workers have suggested that proteoglycans play
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,

Polyamine-Anthracene Conjugates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2679
2 mM glutamine (2 mM), penicillin (100 U/mL), and strepto-
mycin (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 the 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. The same procedure was used
to obtain the data listed in Tables 1 and 2. Initially, the Km
value of spermidine transport was determined in a reaction
volume of 600 µL of Hanks’ 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 incu-
bated at 37 °C for 10 min. The reaction was stopped by adding
3 mL of 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% Triton in distilled water. Two hundred
microliters of the cell lysate was transferred into a 5 mL
scintillation vial containing 3 mL of Pico-Fluor 15. The
respective radioactivity of each sample was measured using a
scintillation counter. The Km value of spermidine transport was
determined by the Lineweaver-Burke analysis as described.48
The ability of conjugates to compete for [14C]-spermidine
uptake was 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 equation49 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.50,51 Cell lines (murine leukemic L1210 cells) were
grown and maintained according to established procedures.7,52
Cells were washed twice in HBSS prior to the transport assay.
P r ep a r a tion of Tr ia m in es 1a -f a n d 6a -c. The synthesis
and full characterization of triamines 1a -f and 6a -c were
reported in the preceding article.13
Gen er a l P r oced u r e for th e N-Boc P r otection (P r ep a -
r a tion of 8). A solution of 7 (5 mmol) in pyridine/methanol
(1:5 v/v, 20 mL) was stirred at 0 °C for 10 min. 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 1 h.
The temperature was then allowed to gradually rise to room
temperature, and the reaction mixture was stirred overnight.
The mixture was evaporated under reduced pressure. The
residue was dissolved in methylene chloride and washed with
deionized water several times. The organic layer was sepa-
rated, dried with anhydrous Na2SO4, filtered, and concen-
trated. The residue could be purified by flash column chro-
matography on silica gel or be used in the next step without
further purification.
Gen er a l P r oced u r e for th e Tosyla tion of th e Hyd r oxy
Gr ou p (P r ep a r a tion of 9). The solution of the N-Boc
protected 8 (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 hour, and 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 decanted off, the
viscous liquid or hemisolid was dissolved in methylene chloride
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 column
chromatography on silica gel or used in the next step without
further purification.
Gen er a l P r oced u r e for th e Su bstitu tion of th e Tosy-
la ted Com p ou n d s w ith th e Am in o Alcoh ols (P r ep a r a tion
of 7) or Dia m in es (P r ep a r a tion of 10). The tosylated
compounds 6 (1 mmol) and amino alcohols (5 mmol) (or
tosylates 9 with diamines) were dissolved in acetonitrile (10
mL), and the mixture was then stirred at 75 °C under N2
overnight. After a check for the disappearance of the tosylate
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 7 (or 10) were used in the next deprotection
step immediately.
Gen er a l P r oced u r e for th e Am in o Gr ou p Dep r otec-
tion (P r ep a r a tion of 11). The N-Boc protected N1-(anthra-
cen-9-ylmethyl)tetraamines (10, 0.5 mmol) were dissolved in
ethanol (5 mL), and the mixture was stirred at 0 °C for 10
min. A 4 N HCl (8 mL) solution was then added dropwise at
0 °C. The reaction mixture was stirred at room temperature
overnight. The solution was then concentrated under reduced
pressure (water bath maintained below 60 °C), and a bright
yellow solid precipitated. The solid was washed several times
with absolute ethanol and provided the pure target compound
11.
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 cells13
were incubated for 3 min with 1d and 11f (10 µM) at 37 °C 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 media and
centrifuged, and the supernatant was removed. The cellular
pellet was resuspended in fresh media, fixed with paraform-
aldehyde, 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.
An th r a cen -9-ylm eth yl[3-(3-h yd r oxy-p r op yla m in o)p r o-
p yl]ca r ba m ic a cid ter t-bu tyl ester (7a ): pale-yellow viscous
liquid; yield 51%; Rf ) 0.53, chloroform/methanol/NH4OH, 90:
1
10:1; H NMR δ 8.39 (s, 1H), 8.37 (d, 2H), 7.95 (d, 2H), 7.43
(m, 4H), 5.42 (br s, 2H), 3.60 (t, 2H), 2.82 (br s, 2H), 2.42 (t,
2H), 2.18 (t, 2H), 1.50 (m, 11H), 1.24 (br s, 2H); 13C NMR δ
155.78, 131.53, 131.44, 129.47, 128.44, 126.61, 125.23, 124.26,
80.24, 64.19, 49.66, 47.29, 43.06, 41.46, 30.98 (2C), 28.95 (3C).
HRMS (FAB) calcd for C26H35N2O3 (M + H)+: 423.2648.
Found: 423.2657.
An th r acen -9-ylm eth yl[3-(4-h ydr oxybu tylam in o)pr opyl]-
ca r ba m ic a cid ter t-b u t yl est er (7b): pale-yellow viscous
liquid; yield 67%; Rf ) 0.38, chloroform/methanol/NH4OH, 90:
1
10:1; H NMR δ 8.40 (s, 1H), 8.38 (d, 2H), 8.00 (d, 2H), 7.45
(m, 4H), 5.50 (br s, 2H), 3.44 (t, 2H), 3.38 (br s, 2H), 2.84 (br
s, 2H), 2.28 (t, 2H), 2.20 (t, 2H), 1.50 (br s, 11H), 1.40 (br s,

2680 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Wang et al.
2H), 1.30 (br s, 2H); 13C NMR δ 155.67, 131.53, 131.44, 129.47,
128.46, 126.64, 125.25, 124.25, 80.34, 62.68, 49.49, 46.86,
43.02, 41.48, 32.62 (2C), 28.94 (3C, CH3), 28.65. HRMS (FAB)
calcd for C27H37N2O3 (M + H)+: 437.2804. Found: 437.2800.
(3C, CH3), 26.31, 26.25, 25.22. HRMS (FAB) calcd for
C
33H46N2O5 Na (M + Na)+: 573.3299. Found: 573.3325.
[5-(An th r a cen -9-ylm eth yl-ter t-bu toxyca r bon yla m in o)-
p en tyl](4-h yd r oxybu tyl)ca r ba m ic a cid ter t-bu tyl ester
(8e): pale-yellow viscous liquid; yield 69%; Rf ) 0.30, hexane/
An th r a cen -9-ylm eth yl[3-(5-h yd r oxyp en tyla m in o)p r o-
p yl]ca r ba m ic a cid ter t-bu tyl ester (7c): pale-yellow viscous
liquid; yield 66%; Rf ) 0.38, chloroform/methanol/NH4OH, 92:
8:1; 1H NMR δ 8.40 (s, 1H), 8.38 (d, 2H), 7.98 (d, 2H), 7.45 (m,
4H), 5.45 (br s, 2H), 3.50 (t, 2H), 2.84 (br s, 2H), 2.25 (t, 2H),
2.20 (t, 2H), 1.50 (br s, 11H), 1.40 (br s, 2H), 1.28 (br s, 4H);
13C NMR δ 155.81, 131.53, 131.44, 129.43, 128.39, 126.54,
125.21, 124.36, 80.09, 62.69, 49.77, 47.55, 43.08, 41.33, 32.79
(2C), 29.84, 28.92 (3C, CH3), 23.73. HRMS (FAB) calcd for
1
acetone, 75:25; H NMR δ 8.42 (s, 1H), 8.39 (d, 2H), 8.00 (d,
2H), 7.50 (m, 4H), 5.50 (br s, 2H), 3.62 (br s, 2H), 3.10 (br s,
2H), 2.85 (br s, 4H), 2.40 (br s, 1H, OH), 1.60 (br s, 9H), 1.50
(m, 4H), 1.34 (br s, 9H), 1.18 (br s, 4H), 0.95 (br s, 2H); 13C
NMR δ 155.80, 131.54, 131.49, 129.41, 128.32, 126.49, 125.20,
124.39, 79.96, 79.39, 62.72, 47.21, 47.01, 44.82, 41.30, 30.07,
28.95 (3C, CH3), 28.82 (3C, CH3), 28.55, 25.27, 24.41 (2C).
HRMS (FAB) calcd for C34H48N2O5 Na (M + Na)+: 587.3455.
Found: 587.3480.
C
28H39N2O3 (M + H)+: 451.2961. Found: 451.2975.
[5-(An th r a cen -9-ylm eth yl-ter t-bu toxyca r bon yla m in o)-
p en tyl](5-h yd r oxyp en tyl)ca r ba m ic a cid ter t-bu tyl ester
(8f): pale-yellow viscous liquid; yield 61%; Rf ) 0.42, hexane/
An th r a cen -9-ylm eth yl[4-(4-h yd r oxybu tyla m in o)bu tyl]-
ca r ba m ic a cid ter t-bu tyl ester (7d ): pale-yellow viscous
liquid; yield 74%; Rf ) 0.36, chloroform/methanol/NH4OH, 92:
8:1; 1H NMR δ 8.42 (s, 1H), 8.39 (d, 2H), 8.01 (d, 2H), 7.50 (m,
4H), 5.50 (br s, 2H), 3.50 (t, 2H), 2.80 (br s, 2H), 2.40 (t, 2H),
2.20 (t, 2H), 1.58 (br s, 13H), 1.24 (m, 2H), 1.10 (m, 4H); 13C
NMR δ 155.77, 131.54, 131.49, 129.43, 128.35, 126.56, 125.24,
124.37, 80.12, 62.84, 49.66, 48.99, 44.70, 41.46, 32.98, 29.18,
28.95 (3C, CH3), 26.99, 26.46. HRMS (FAB) calcd for C28H39N2O3
(M + H)+: 451.2961. Found: 451.2947.
1
acetone, 70:30; H NMR δ 8.42 (s, 1H), 8.39 (d, 2H), 8.00 (d,
2H), 7.50 (m, 4H), 5.50 (br s, 2H), 3.62 (br s, 2H), 3.10-2.80
(m, 6H), 1.64 (m, 11H, 3CH3 + CH2), 1.40 (m, 11H, 3CH3
+
CH2), 1.25 (m, 6H), 0.90 (br s, 2H); 13C NMR δ 155.71, 131.55,
131.50, 129.41, 128.31, 126.48, 125.21, 124.41, 79.94, 79.38,
63.04, 47.20, 47.00, 44.81, 41.28, 32.71, 28.94 (3C, CH3), 28.83
(3C, CH3), 28.54, 28.49, 28.26, 24.43, 23.24. HRMS (FAB) calcd
for C35H51N2O5 (M + H)+: 579.3798. Found: 579.3780.
Tolu en e-4-su lfon ic a cid 4-{[3-(a n th r a cen -9-ylm eth yl-
ter t-bu toxyca r bon yla m in o)p r op yl]-ter t-bu toxyca r bon yl-
a m in o}bu tyl ester (9b): pale-yellow viscous liquid; yield
70%; Rf ) 0.35, hexane/acetone, 75:25; 1H NMR δ 8.42 (s, 1H),
8.39 (d, 2H), 8.00 (d, 2H), 7.78 (d, 2H), 7.48 (m, 4H), 7.28 (d,
2H), 5.50 (br s, 2H), 3.90 (t, 2H), 2.82 (br s, 2H), 2.60 (br s,
4H), 2.40 (s, 3H), 1.58 (br s, 9H, 3CH3), 1.42 (br s, 2H), 1.22
(m, 13H, 3CH3 + 2CH2); 13C NMR δ 155.19, 144.87, 133.28,
131.57, 131.43, 130.03, 129.46, 128.42, 128.05, 126.67, 125.29,
124.18, 80.19, 79.38, 70.51, 45.26, 44.71, 43.27, 41.78, 28.95
(3C, CH3), 28.80, 28.64 (3C, CH3), 26.46, 24.20, 22.03. HRMS
(FAB) calcd for C39H51N2O7S (M + H)+: 691.3417. Found:
691.3411.
An th r acen -9-ylm eth yl[5-(4-h ydr oxybu tylam in o)pen tyl]-
ca r ba m ic a cid ter t-bu tyl ester (7e): pale-yellow viscous
liquid; yield 90%; Rf ) 0.66, chloroform/methanol/NH4OH, 92:
8:2; 1H NMR δ 8.42 (s, 1H), 8.40 (d, 2H), 8.01 (d, 2H), 7.51 (m,
4H), 5.55 (br s, 2H), 3.50 (t, 2H), 2.80 (br s, 2H), 2.52 (t, 2H),
2.28 (t, 2H), 1.58 (br s, 13H), 1.20 (m, 4H), 0.88 (br s, 2H); 13
C
NMR δ 155.91, 131.53, 131.44, 129.42, 128.32, 126.51, 125.22,
124.43, 79.95, 62.86, 49.84, 49.42, 44.86, 41.41, 33.06, 29.35,
29.30, 28.96 (3C), 28.52, 24.70. HRMS (FAB) calcd for
C
29H41N2O3 (M + H)+: 465.3112. Found: 465.3118.
An th r a cen -9-ylm eth yl[5-(5-h yd r oxyp en tyla m in o)p en -
tyl]ca r ba m ic a cid ter t-bu tyl ester (7f): pale-yellow viscous
liquid; yield 85%; Rf ) 0.39, chloroform/methanol/NH4OH, 90:
1
10:1; H NMR δ 8.42 (s, 1H), 8.40 (d, 2H), 8.00 (d, 2H), 7.50
Tolu en e-4-su lfon ic a cid 4-{[4-(a n th r a cen -9-ylm eth yl-
ter t-b u t oxyca r b on yla m in o)bu t yl]-ter t-b u t oxyca r b on yl-
a m in o}bu tyl ester (9d ): pale-yellow viscous liquid; yield
74%; Rf ) 0.43, hexane/acetone, 75:25; 1H NMR δ 8.42 (s, 1H),
8.39 (d, 2H), 8.00 (d, 2H), 7.78 (d, 2H), 7.48 (m, 4H), 7.28 (d,
2H), 5.50 (br s, 2H), 4.00 (t, 2H), 2.88 (br s, 2H), 2.80-2.62
(m, 4H), 2.40 (s, 3H), 1.58 (br s, 9H, 3CH3), 1.38 (m, 13H,
3CH3 + 2CH2), 1.05 (br s, 4H); 13C NMR δ 155.43, 144.89,
133.30, 131.56, 131.49, 130.04, 129.47, 128.39, 128.06, 126.55,
125.23, 124.33, 80.10, 79.42, 70.55, 46.79, 46.20, 44.74, 41.46,
28.95 (3C, CH3), 28.74 (3C, CH3), 26.57 (2C), 26.22, 24.51,
22.02.
(m, 4H), 5.52 (br s, 2H), 3.60 (t, 2H), 2.80 (br s, 2H), 2.48 (t,
2H), 2.28 (t, 2H), 1.90 (br s, 2H, NH, OH), 1.50 (br s, 11H),
1.45 (m, 4H), 1.22 (m, 2H), 1.19 (m, 2H), 0.92 (m, 2H); 13C NMR
δ 155.96, 131.53, 131.44, 129.40, 129.08, 128.31, 126.47,
125.19, 124.41, 79.91, 62.73, 49.97 (2C), 44.86, 41.31, 32.78,
29.92, 29.70, 28.94 (3C), 28.58, 24.83, 23.81. HRMS (FAB) calcd
for C30H43N2O3 (M + H)+: 479.3274. Found: 479.3268.
[3-(An th r a cen -9-ylm eth yl-ter t-bu toxyca r bon yla m in o)-
p r op yl](3-h yd r oxyp r op yl)ca r ba m ic a cid ter t-bu tyl ester
(8a ): pale-yellow viscous liquid; yield 68%; Rf ) 0.38, hexane/
acetone, 3:1; 1H NMR δ 8.42 (s, 1H), 8.39 (d, 2H), 8.00 (d, 2H),
7.50 (m, 4H), 5.50 (br s, 2H), 3.70 (br s, 1H, OH), 3.30 (br s,
2H), 2.85 (br s, 2H), 2.78 (br s, 2H), 2.60 (br s, 2H), 1.60 (br s,
9H), 1.28 (br s, 13H). HRMS (FAB) calcd for C31H43N2O5 (M +
H)+: 523.3172. Found: 523.3183.
N1-{3-[3-(An th r a cen -9-ylm eth yl)a m in o]p r op yla m in o}-
bu ta n e-1,4-d ia m in e tetr a h yd r och lor id e (11a ): bright-
1
yellow solid; yield 96%; H NMR δ 8.72 (s, 1H), 8.28 (d, 2H),
8.16 (d, 2H), 7.64 (t, 2H), 7.58 (t, 2H), 5.20 (br s, 2H), 3.38 (br
s, 2H), 3.00 (m, 8H), 2.84 (br s, 2H), 2.12 (br s, 2H), 2.00 (br s,
2H), 1.62 (br s, 4H); 13C NMR δ 130.73, 130.54, 130.17, 129.55,
127.82, 125.59, 122.57, 120.17, 47.38, 44.91 (2C), 44.83, 44.70,
43.19, 39.11, 24.25, 23.09, 22.99 (2C). HRMS (FAB) calcd for
[3-(An th r a cen -9-ylm eth yl-ter t-bu toxyca r bon yla m in o)-
p r op yl](4-h yd r oxybu tyl)ca r ba m ic a cid ter t-bu tyl ester
(8b): pale-yellow viscous liquid; yield 82%; Rf ) 0.38, hexane/
1
acetone, 70:30; H NMR δ 8.42 (s, 1H), 8.39 (d, 2H), 8.00 (d,
25H37N4 (M + H - 4HCl)+: 393.3013. Found: 393.3020.
N-(3-Am in opr opyl)-N-{3-[(an th r acen -9-ylm eth yl)am in o]-
2H), 7.50 (m, 4H), 5.50 (br s, 2H), 3.56 (br s, 2H), 2.84 (br s,
2H), 2.70 (br s, 4H), 2.50 (br s, 1H, OH), 1.60 (br s, 9H), 1.28
(br s, 15H); 13C NMR δ 155.50, 155.44, 131.57, 131.46, 129.47,
128.86, 128.42, 126.66, 125.27, 124.25, 80.15, 79.34, 62.73,
46.06, 44.89, 43.21, 41.70, 29.82, 28.95 (3C, CH3), 28.68 (3C,
CH3), 28.04, 24.74. HRMS (FAB) calcd for C32H45N2O5 (M +
H)+: 537.3328. Found: 537.3338.
C
p r op yl}b u t a n e-1,4-d ia m in e t et r a h yd r och lor id e (11b ):
bright-yellow solid; yield 91%; 1H NMR δ 8.73 (s, 1H), 8.28 (d,
2H), 8.17 (d, 2H), 7.64 (t, 2H), 7.58 (t, 2H), 5.18 (s, 2H), 3.30
(t, 2H), 2.96 (m, 10H), 2.10 (m, 2H), 1.90 (m, 2H), 1.60 (br s,
4H); 13C NMR δ 130.70, 130.51, 130.13, 129.51, 127.77, 125.55,
122.54, 120.15, 47.25 (2C), 44.81 (2C), 44.71, 43.12, 36.8, 24.06,
23.05 (2C), 22.97. HRMS (FAB) calcd for C25H37N4 (M + H -
4HCl)+: 393.3013. Found: 393.3018.
N-(4-Am in obu tyl)-N-{3-[(a n th r a cen -9-ylm eth yl)a m in o]-
p r op yl}b u t a n e-1,4-d ia m in e t et r a h yd r och lor id e (11c):
bright-yellow solid; yield 95%; 1H NMR δ 8.80 (s, 1H), 8.40 (d,
2H), 8.19 (d, 2H), 7.66 (t, 2H), 7.60 (t, 2H), 5.21 (s, 2H), 3.40
(t, 2H), 3.02 (t, 2H), 2.92 (m, 6H), 2.82 (t, 2H), 2.11 (m, 2H),
1.62 (m, 8H); 13C NMR δ 130.85, 130.62, 130.29, 129.57, 127.86,
[4-(An th r a cen -9-ylm eth yl-ter t-bu toxyca r bon yla m in o)-
bu tyl](4-h yd r oxybu tyl)ca r ba m ic a cid ter t-bu tyl ester
(8d ): pale-yellow viscous liquid; yield 71%; Rf ) 0.43, hexane/
1
acetone, 70:30; H NMR δ 8.42 (s, 1H), 8.39 (d, 2H), 8.00 (d,
2H), 7.50 (m, 4H), 5.50 (br s, 2H), 3.62 (t, 2H), 3.00 (br s, 2H),
2.82 (br s, 4H), 2.02 (br s, 1H, OH), 1.60 (br s, 9H), 1.50 (m,
6H), 1.34 (br s, 9H), 1.18 (br s, 2H); 13C NMR δ 155.64, 131.56,
131.49, 129.46, 128.38, 126.54, 125.21, 124.33, 80.08, 79.38,
62.83, 46.99, 46.97, 44.71, 41.48, 29.96, 28.95 (3C, CH3), 28.78

Polyamine-Anthracene Conjugates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2681
(2) Wang, L.; Price, H. L.; J uusola, J .; Kline, M.; Phanstiel, O., IV.
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) 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 (10), 1984-1990.
(4) 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 (28), 17604-17609.
(5) Bergeron, R. J .; Feng, Y.; Weimar, W. R.; McManis, J . S.;
Dimova, H.; Porter, C.; Raisler, B.; Phanstiel, O. A Comparison
of Structure-Activity Relationships between Spermidine and
Spermine Analogue Antineoplastics. J . Med. Chem. 1997, 40,
1475-1494.
(6) 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 (10), 717-
729.
(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 analogs. J . Cell. Physiol. 1993,
155, 399-407.
125.64, 122.61, 120.38, 47.26, 47.18, 47.10, 44.86, 44.72, 43.25,
39.06, 24.23, 23.07 (2C), 22.99. HRMS (FAB) calcd for C26H39N4
(M + H - 4HCl)+: 407.3175. Found: 407.3165.
N-(4-Am in obu tyl)-N-{3-[(a n th r a cen -9-ylm eth yl)a m in o]-
p r op yl}p en t a n e-1,5-d ia m in e t et r a h yd r och lor id e (11d ):
bright-yellow solid; yield 88%; 1H NMR δ 8.70 (s, 1H), 8.28 (d,
2H), 8.15 (d, 2H), 7.63 (t, 2H), 7.58 (t, 2H), 5.20 (s, 2H), 3.38
(t, 2H), 3.01 (t, 2H), 2.90 (m, 8H), 2.11 (m, 2H), 1.62 (br s,
8H), 1.28 (m, 2H); 13C NMR δ 130.71, 130.52, 130.14, 129.53,
127.80, 125.57, 122.55, 120.16, 47.70, 47.54, 47.12, 44.84,
44.65, 43.13, 39.10, 25.39 (2C), 24.26, 23.16, 23.08, 22.97.
HRMS (FAB) calcd for C27H41N4 (M + H - 4HCl)+: 421.3326.
Found: 421.3327.
N-[4-(3-Am in op r op yla m in o)b u t yl]-N-a n t h r a cen -9-yl-
m et h ylb u t a n e-1,4-d ia m in e t et r a h yd r och lor id e (11e):
bright-yellow solid; yield 94%; 1H NMR δ 8.79 (s, 1H), 8.40 (d,
2H), 8.19 (d, 2H), 7.68 (t, 2H), 7.60 (t, 2H), 5.21 (s, 2H), 3.28
(t, 2H), 2.92 (m, 10H), 1.92 (m, 2H), 1.80 (m, 2H), 1.70 (br s,
6H); 13C NMR δ 130.69, 130.41, 130.09, 129.49, 127.73, 125.53,
122.52, 120.37, 47.28, 47.20, 47.12, 47.06, 44.81, 42.82, 36.82,
24.05, 23.15, 23.08 (2C), 23.01. HRMS (FAB) calcd for C26H39N4
(M + H - 4HCl)+: 407.3169. Found: 407.3166.
N -[4-(4-Am in ob u t yla m in o)b u t yl]-N -a n t h r a ce n -9-yl-
m eth ylbu tan e-1,4-diam in e tetr ah ydr och lor ide (11f): bright-
(8) Kashiwagi, K.; Endo, H.; Kobayashi, H.; Takio, K.; Igarashi, K.
Spermidine-preferential uptake systemin Escherichia coli. ATP
hydrolysis by PotA protein and its association with membrane.
J . Biol. Chem. 1995, 270, 25377-25382.
(9) 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.
1
yellow solid; yield 86%; H NMR δ 8.79 (s, 1H), 8.40 (d, 2H),
8.19 (d, 2H), 7.68 (t, 2H), 7.60 (t, 2H), 5.21 (s, 2H), 3.24 (br s,
2H), 2.92 (br s, 8H), 2.84 (br s, 2H), 1.80 (br s, 2H), 1.64 (br s,
10H); 13C NMR δ 130.75, 130.46, 130.14, 129.53, 127.77,
125.57, 122.57, 120.45, 47.24, 47.20, 47.14 (2C), 47.08, 42.88,
39.09, 24.26, 23.18, 23.12 (2C), 23.10, 23.06. HRMS (FAB) calcd
for C27H41N4 (M + H - 4HCl)+: 421.3326. Found: 421.3310.
N-[4-(3-Am in op r op yla m in o)b u t yl]-N-a n t h r a cen -9-yl-
m eth ylp en ta n e-1,5-d ia m in e tetr a h yd r och lor id e (11g):
bright-yellow solid; yield 88%; 1H NMR δ 8.79 (s, 1H), 8.40 (d,
2H), 8.19 (d, 2H), 7.66 (t, 2H), 7.60 (t, 2H), 5.20 (s, 2H), 3.22
(t, 2H), 2.90 (m, 10H), 1.92 (m, 2H), 1.66 (m, 8H), 1.40 (m,
2H); 13C NMR δ 130.70, 130.36, 130.07, 129.49, 127.72, 125.54,
122.53, 120.50, 47.64, 47.53, 47.30, 47.05, 44.82, 42.69, 36.84,
25.36, 25.28, 24.07, 23.27, 23.11, 23.09. HRMS (FAB) calcd
for C27H41N4 (M + H- 4HCl)+: 421.3326. Found: 421.3319.
N -[4-(4-Am in ob u t yla m in o)b u t yl]-N -a n t h r a ce n -9-yl-
m eth ylp en ta n e-1,5-d ia m in e tetr a h yd r och lor id e (11h ):
(10) 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.
(11) (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 analogs 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.
(12) 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.
1
bright-yellow solid; yield 92%; H NMR δ 8.78 (s, 1H,), 8.39
(d, 2H), 8.18 (d, 2H), 7.66 (t, 2H), 7.60 (t, 2H), 5.20 (s, 2H),
3.22 (t, 2H), 2.88 (br s, 8H), 2.80 (m, 2H), 1.74 (m, 2H), 1.62
(br s, 10H), 1.40 (m, 2H); 13C NMR δ 130.70, 130.36, 130.07,
129.49, 127.72, 125.54, 122.53, 120.50, 47.64, 47.53, 47.20,
47.14, 47.06, 42.70, 39.08, 25.36, 25.28, 24.24, 23.27, 23.12
(2C), 23.08. HRMS (FAB) calcd for C28H43N4 (M + H -
4HCl)+: 435.3482. Found: 435.3477.
(13) Wang, C.; Delcros, J .-G.; Biggerstaff, J .; Phanstiel, O., IV.
Synthesis and Biological Evaluation of N1-(Anthracen-9-ylmeth-
yl)triamines as Molecular Recognition Elements for the Polyamine
Transporter. J . Med. Chem. 2003, 46, 2663-2671.
N-[5-(4-Am in ob u t yla m in o)p en t yl]-N-a n t h r a cen -9-yl-
m eth ylp en ta n e-1,5-d ia m in e tetr a h yd r och lor id e (11i):
bright-yellow solid; yield 99%; 1H NMR δ 8.70 (s, 1H), 8.28 (d,
2H), 8.13 (d, 2H), 7.66 (t, 2H), 7.56 (t, 2H), 5.24 (s, 2H), 3.16
(t, 2H), 2.88 (m, 10H), 1.70 (m, 2H), 1.58 (m, 10H), 1.32 (m,
4H); 13C NMR δ 130.69, 130.35, 130.06, 129.49, 127.70, 125.52,
122.52, 120.50, 47.62, 47.56, 47.48, 47.44, 47.11, 42.67, 39.08,
25.42 (2C), 25.34, 25.27, 24.25, 23.27, 23.21, 23.07. HRMS
(FAB) calcd for C29H45N4 (M + H - 4HCl)+: 449.3644. Found:
449.3650.
(14) Mandel, J . L.; Flintoff, W. F. Isolation of mutant mammalian
cells altered in polyamine transport. J . Cell. Physiol. 1978, 97,
335-344.
(15) 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.
(16) 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.
(17) 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.
(18) 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.
(19) Cai, J .; Soloway, A. H. Synthesis of Carboranyl Polyamines for
DNA Targeting. Tetrahedron Lett. 1996, 37, 9283-9286.
(20) 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 thank the Chinese Scholarship
Council for partial support of C.W. The assistance of
Dr. Richard Gardner and Mr. Tito Carias in obtaining
the deconvolution microscopy images is also appreciated.
(21) Blagbrough, I. S.; Geall, A. J . Homologation of Polyamines in
the Synthesis of Lipo-Spermine Conjugates and Related Li-
poplexes. Tetrahedron Lett. 1998, 443-446.
(22) Cullis, P. M.; Merson-Davies, L.; Weaver, R. Mechanism and
reactivity of chlorambucil and chlorambucil-spermidine conju-
gate. J . Am. Chem. Soc. 1995, 117, 8033-8034.
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.

2682 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Wang et al.
(23) 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.
(24) Blagbrough, I. S.; Geall, A. J . Practical Synthesis of Unsym-
metrical Polyamine Amides. Tetrahedron Lett. 1998, 439-442.
(25) 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.
(26) 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.
(27) 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 (6), 861-872.
(28) 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.
(29) 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.
(30) 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.
(38) 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.
(39) 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 analog
recognition. J . Med. Chem. 1995, 38, 2278-2285.
(40) 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.
(41) Xia, C. Q.; Yang, J . J .; Ren, S.; Lien, E. J . QSAR analysis of
polyamine transport inhibitors in L1210 cells. J . Drug Targeting
1998, 6 (1), 65-77.
(42) O’Sullivan, M. C.; Golding, B. T.; Smith, L. L.; Wyatt, I.
Molecular features necessary for the uptake of diamines and
related compounds by the polyamine receptor of rat lung slices.
Biochem. Pharmacol. 1991, 41, 1839-1848.
(43) Kuksa, V.; Buchan, R.; Lin, P. K. T. Synthesis of Polyamines,
Their Derivatives, Analogues and Conjugates. Synthesis 2000,
No. 9, 1189-1207.
(44) Alhonen-Hongisto, L.; Seppanen, P.; J anne, J . Intracellular
putrescine and spermidine deprivation induces increased uptake
of the natural polyamines and methylglyoxal bis(guanylhydra-
zone). Biochem. J . 1980, 192, 941-945.
(45) Minchin, R. F.; Raso, A.; Martin, R. L.; Ilett, K. F. Evidence for
the existence of distinct transporters for the polyamines pu-
trescine and spermidien in B16 melanoma cells. Eur. J . Biochem.
1991, 200, 457-462.
(46) Rinehart, C. A. J .; Chen, K. Y. Characterization of the polyamine
transport system in mouse neuroblastoma cells. Effects of
sodium and system A amino acids. J . Biol. Chem. 1984, 259,
4750-4756.
(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 eryth-
roleukemia cells. Biochem. J . 1995, 312, 933-938.
(49) 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.
(50) 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.
(51) 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.
(52) 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.
(31) 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, 3464-3476.
(32) Belting, M.; Persson, S.; Fransson, L.-A. Proteoglycan involve-
ment in polyamine uptake. Biochem. J . 1999, 338, 317-323.
(33) (a) Schindler, M.; Koppel, D. E.; Sheetz, M. P. Modulation of
membrane protein lateral mobility by polyphosphates and
polyamines. Proc. Natl. Acad. Sci. U.S.A. 1980, 77 (3), 1457-
1461. (b) Ballas, S. K.; Mohandas, N.; Marton, L. J .; Shohet, S.
B. Stabilization of erythrocyte mebranes by polyamines. Proc.
Natl. Acad. Sci. U.S.A. 1983, 80 (7), 1942-1946. (c) Schuber, F.
Influence of polyamines on membrane functions. Biochem. J .
1989, 269, 1-10.
(34) 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. Biochem. J . 2002, 367, 347-357.
(35) Fransson, L.-A. Glypicans. Int. J . Biochem. Cell Biol. 2002, 1348,
1-5.
(36) 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.
(37) 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.
J M020598G