Amino acid/spermine conjugates: Polyamine amides as potent spermidine uptake inhibitors

Article abstract of DOI:10.1021/jm0101040

In this paper we describe the synthesis and characterization of a series of simple spermine/amino acid conjugates, some of which potently inhibit the uptake of spermidine into MDA-MB-231 breast cancer cells. The presence of an amide in the functionalized

Full text of DOI:10.1021/jm0101040

3632
J . Med. Chem. 2001, 44, 3632-3644
Am in o Acid /Sp er m in e Con ju ga tes: P olya m in e Am id es a s P oten t Sp er m id in e
Up ta k e In h ibitor s
Mark R. Burns,* C. Lance Carlson, Scott M. Vanderwerf, J osh R. Ziemer, Reitha S. Weeks, Feng Cai,
Heather K. Webb, and Gerard F. Graminski
Oridigm Corporation, 4010 Stone Way North, Suite 220, Seattle, Washington 98103
Received March 7, 2001
In this paper we describe the synthesis and characterization of a series of simple spermine/
amino acid conjugates, some of which potently inhibit the uptake of spermidine into MDA-
MB-231 breast cancer cells. The presence of an amide in the functionalized polyamine appeared
to add to the affinity for the polyamine transporter. The extensive biological characterization
of an especially potent analogue from this series, the Lys-Spm conjugate (31), showed this
molecule will be an extremely useful tool for use in polyamine research. It was shown that the
use of 31 in combination with DFMO led to a cytostatic growth inhibition of a variety of cancer
cells, even when used in the presence of an extracellular source of transportable spermidine.
It was furthermore shown that this combination effectively reduced the cellular levels of
putrescine and spermidine while not affecting the levels of spermine. These facts together with
the nontoxic nature of 31 make it a novel lead for further anticancer development.
In tr od u ction
mechanism-based inhibitor of ODC,22 causes a substan-
tial depletion of intracellular 1 and 2 with resultant cell
growth inhibition. Upon supplementing the culture
medium of these DFMO-treated cells with exogenous
polyamines, transport activity rose severalfold.23,24 The
resulting increase in intracellular polyamine levels
restored the cells to their original rate of growth.
Decades of research on the biological activities that
the polyamines putrescine (1), spermidine (2), and
spermine (3) play in cellular processes1 have shown the
profound role they play in life. As polycations at physi-
ological pH, they bind tightly to and strongly modulate
the biological activities of anionic cellular components.2-10
Due to these and other findings, many researchers have
explored the effect of synthetic modifications of the
polyamine structure in the pursuit of chemotherapeutic
agents.11
Numerous multidisciplinary studies have shown that
intracellular concentrations of the polyamines are highly
regulated at many steps in their biosynthesis, catabo-
lism, and transport. The fact that the cell contains such
a complex apparatus for the tight control of the levels
of these molecules indicates that only a very narrow
window of concentration is tolerated. Ornithine decar-
boxylase (ODC), the rate-limiting enzyme in polyamine
biosynthesis, catalyzes the production of 1 from its
precursor ornithine. An increase in the activity of ODC
has been associated with tumor growth.12-14 Polyamine
transport into mammalian cells is energy and temper-
ature dependent, saturable, and carrier mediated and
Several experimental lines of evidence support the
conclusion that increased effectiveness of ODC inhibi-
tion can be obtained by interfering with the polyamine
transport apparatus. A mutant L1210 leukemia cell line
was shown to have greatly reduced polyamine transport
activity following selection for resistance to methylgly-
coxal bis(guanylhydrazone) (MGBG), a cytotoxic AdoMet-
DC inhibitor that is taken up by the same transport
system as the polyamines. Mice inoculated with these
cells had a much greater response to DFMO treatment
(87% increase in median survival time, 13 of 40 mice
cured) than mice inoculated with the parental cell line
(22% increase in median survival time).25 A second
experimental approach is based on the fact that the
microbial flora in the gastrointestinal tract produces a
significant source of extracellular polyamines.26 When
this source of polyamines is removed by antibiotic
treatment, DFMO’s previously moderate growth inhibi-
tory effects on Lewis lung carcinoma or L1210 cells are
markedly potentiated.27 Finally, an additional source of
polyamines is from the diet.28 By feeding a polyamine-
free diet to DFMO-treated nude mice, MCF-7 human
breast cancer xenografts contained greatly reduced
levels of putrescine in comparison to DFMO treatment
alone.29 In additional animal models, complete polyamine
deprivation also enhanced DFMO’s growth inhibitory
effectiveness.30-32
operates against
a substantial concentration gra-
dient.15-17 Ample experimental proof exists that poly-
amine concentration homeostasis is aided by this trans-
port system. Changes in the requirements for polyamines
in response to growth stimulation are reflected by
increases in the transport activity. Stimulation of hu-
man fibroblasts to proliferate by serum or epidermal
growth factor leads to an 18-100-fold increase in the
uptake of putrescine.18,19 Tumors have been shown to
have an increased rate of putrescine uptake.20,21 Inhibi-
tion of polyamine biosynthesis in cells in culture by
R-(difluoromethyl)ornithine (DFMO), a well-studied
Given the importance of extracellular sources of
polyamines to the growth of cancer, pharmacological
agents that block polyamine transport are desired. In
this paper we address these needs by describing the
* To whom correspondence should be addressed. Phone: (206) 675-
8880. Fax: (206) 675-8881. E-mail: markburns@oridigm.com.
10.1021/jm0101040 CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/15/2001

Amino Acid/ Spermine Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3633
Sch em e 1. Synthesis of Amino Acid/Spermine Conjugatesa
a
+
Reagents and conditions: (a) MeOH (for 4-NO2Ph esters) or CH2Cl2 (for N-hydroxysuccinimide esters); (b) BioRex 70 (NH4 form)
t
cation-exchange chromatography; (c) 3 N HCl in MeOH (protecting group (PG) ) Boc) or H2, Pd(OH)2, EtOH/H2O (PG ) Cbz).
design, synthesis, and biological evaluation of a series
of simple amino acid/spermine conjugates that act as
potent polyamine transport inhibitors in the MDA-MB-
231 human breast cancer cell line. These compounds
were evaluated on the basis of their (1) ability to inhibit
the uptake of radiolabeled spermidine into MDA-MB-
231 breast cancer cells, (2) their ability to increase the
growth inhibitory effects of DFMO on MDA-MB-231
cells in culture even in the presence of 1 µM extracel-
lular spermidine, (3) their inability to rescue cells from
the growth inhibitory effects of DFMO in the absence
of extracellular polyamines, and (4) their ability to
deplete the intracellular levels of polyamines after
combination treatment with DFMO. Finally, these
compounds have limited cytotoxic properties when used
alone, thus increasing the potential of providing tumor
selectivity.
salts. All novel analogues gave satisfactory analysis by
1
TLC, H and 13C NMR, and HRMS by MALDI-FTMS.
Resu lts
Ki Deter m in a tion . Each compound was evaluated
3
for its ability to inhibit the uptake of H-radiolabeled
spermidine into MDA-MB-231 cells. Ki values are shown
in Tables 1 and 2. All compounds described in this paper
displayed competitive kinetics versus spermidine up-
take. The Km values for putrescine, spermidine, and
spermine are included in Table 1 for comparative
purposes. On the basis of biological screening of a
variety of synthetic and natural products, the J oro
spider toxin J STx-3 (4)37 and a simplified synthetic
analogue, (1-naphthylacetyl)spermine (NpAc-Spm, 5),38
(Table 1) were both discovered to be effective transport
inhibitors with Ki values of 190 and 178 nM, respec-
tively. An additional, relatively complex polyamine
natural product, 15-deoxyspergualin (DSQ, 6),39 was
also shown to have moderate activity as a polyamine
uptake inhibitor in MDA-MB-231 cells (Ki ) 81 nM).
Comparison of these Ki values with the apparent Km
values determined for 1 (1280 ( 340 nM), 2 (113 ( 30
nM), or 3 (280 ( 60 nM) support the view that these
polyamine analogues may bind with equal or greater
affinity to the polyamine transport apparatus. It is
unclear at present whether this property has any role
in the pharmacological activities of these natural prod-
ucts.
Simple polyamine amides were equally effective at
inhibition of spermidine uptake. Nonfunctionalized acyl
groups were placed on the N1 position of spermine to
give analogues 7-13 (Table 1). An analogue with a low
Ki value in this subset was N1-Boc-spermine (9) (Ki )
86 nM), a molecule described in the chemical literature
as a protected polyamine intermediate.40 It is interesting
to note that N1-acetylspermine (7) had an activity (Ki =
177 nM) similar to that of the substrate 2 (Km ) 113 (
30 nM). It appears that the presence of a hydrophobic
propylacetyl amide moiety does not harm the affinity
compared to the unsubstituted spermidine structure. It
is also interesting to note the loss of activity in making
the subtle change of removal of one methylene group
in the polyamine, exemplified by comparing 7 (Ki ) 177
Syn th etic Ch em istr y
Several reports describe the selective primary mono-
substitution of the polyamines.33,34 Typically, an excess
of the polyamine is treated over time with the acylating
reagent. Presumably, the higher nucleophilicity of the
secondary amines is masked by the greater steric
hindrance of these amines, thus giving a higher yield
of the monosubstituted primary amide products. 4-Ni-
trophenyl or N-hydroxysuccinyl active esters of the
variously protected (N-tBoc or N-Cbz) amino acids were
either purchased from commercial sources or synthe-
sized using standard procedures from the free acid
derivatives.35 The activated amino esters were coupled
directly to spermine (1.2 equiv) to yield a mixture of
mono- and disubstituted products together with unre-
acted spermine (Scheme 1). The protected crude mix-
tures were effectively separated using chromatography
over BioRex 70 (NH4+ form) cation-exchange resin with
a linear gradient of 0-2 N NH4OH in H2O.36 The more
lipophilic, protected amino acid derivatives (e.g., 11, 12,
or 31) required the inclusion of MeOH (25%) in the
eluting buffer to ensure complete solution during chro-
matography. After this purification, the protecting
groups were removed under standard conditions and the
desired conjugates were obtained as their hydrochloride

3634 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Burns et al.
Ta ble 1. Km or Ki Values for [3H]Spermidine Transport into MDA-MB-231 Cellsa
a
Ki values are the average of two independent determinations using MDA-MB-231 cells unless otherwise noted. Repeat determinations
b
were generally within 25% of each other. See the Experimental Section for the procedure. One Ki determination.

Amino Acid/ Spermine Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3635
Ta ble 2. Ki and EC50 Values for Amino Acid/Spermine Conjugates

3636 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Ta ble 2. (Continued)
Burns et al.
a
Ki values are the average of two independent determinations using MDA-MB-231 cells unless otherwise noted. Repeat determinations
b
were generally within 25% of each other. See the Experimental Section for the procedure. EC50 values were determined using a 6 day
assay of MDA-MB-231 cells in the presence of 230 µM DFMO, 1 mM AG, and 1.0 µM spermidine.
nM) with N1-acetylnorspermine (N1-Ac-3,3,3, 10) (Ki )
>1000 nM). Insights into the steric environment of this
binding domain can be seen by comparison of amides
11 and 12. The bulky adamantyl group is better toler-
ated if a methylene spacer is included between the
polycyclic moiety (11, Ki ) 256 nM, versus 12, Ki ) 672
nM).
Further increases in transport inhibition potency are
obtained by placing an amino group at the end of the
acyl group. The glycine (14) and â-alanine (15) conju-
gates showed Ki values of 87 and 65 nM, respectively
(Table 2). Given the gain in spermidine transport
inhibition activity shown by the amino-containing gly-
cine/spermine conjugate (14) as compared to 7 (87 nM

Amino Acid/ Spermine Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3637
versus 177 nM), we next examined the activities of a
more extensive series of R-amino acid/spermine conju-
gates. A series of aliphatic hydrocarbon substituted
amino acids was produced. All amino acid derivatives
are the L-stereoisomers unless otherwise noted. R-Meth-
yl substitution had a negative impact on the activity
(compare Gly-Spm (14) (Ki ) 87 nM) with R-Ala-Spm
(16) (Ki ) 144 nM) or Aib-Spm (17) (Ki ) 191 nM)).
Comparison of the L-Val-Spm conjugate (18) (Ki ) 84
nM) with the D-Val-Spm conjugate (19) (Ki ) 58 nM)
showed only a minor stereochemical preference for
activity. Notable steric effects were suggested by subtle
changes in the placement of alkyl groups on the side
chain. Addition of an extra methyl group to the Val side
chain reduced the activity (tButGly-Spm, 20; Ki ) 127
nM). Placement of an extra methyl group at the termi-
nal methyl of the Val conjugate again reduced the
activity as a transport inhibitor (Ile-Spm, 21; Ki ) 150
nM). By simply moving this methyl group one position,
an improvement in activity was observed (Leu-Spm, 22;
Ki ) 46 nM). Increased steric bulk on this derivative
again reduced the activity (Cha-Spm, 23; Ki ) 196 nM).
Formation of a ring by coupling proline with spermine
produced a higher activity analogue (Pro-Spm, 24; Ki
) 38 nM).
We next explored the effects of using functionalized
amino acids as conjugates with spermine. The aspar-
agine/spermine (25) (Ki ) 95 nM) and glutamine/
spermine (26) (Ki ) 177 nM) conjugates were moder-
ately good inhibitors. The Met-Spm (27) (Ki ) 65 nM),
Ser-Spm (28) (Ki ) 55 nM), Orn-Spm (30) (Ki ) 47 nM),
and Lys-Spm (31) (Ki ) 32 nM) conjugates were all very
good inhibitors and had Ki values substantially below
100 nM. The Thr-Spm conjugate (29) was a less effective
inhibitor (Ki ) 213 nM).
F igu r e 1. Growth inhibition of MDA-MB-231 cells with 31
and DFMO. MDA-MB-231 cells were incubated with 0.50 µM
SPD, 1.0 mM AG, and 0.1-100 µM 31 ( 1.0 mM DFMO during
a 6-day growth assay. There was no growth inhibition with
1.0 mM DFMO and 0.50 µM SPD. The cell number was
determined by MTS/PMS assay from triplicate wells. The bars
represent the standard deviation from triplicate wells.
showed an effective EC50 of 3 µM. Other analogues that
had notable activity in this assay included the stereo-
chemical pairs of the D- versus L-Trp/spermine and the
D- versus L-Val/spermine conjugates. It is interesting to
note that 33 gave a better EC50 compared with 32. This
slight increase in activity of the D-stereoisomers, as
reflected by both the Ki and the EC50 values, was
repeated with the Val-Spm pairs (19 more active than
18).
In several cases there appears to be limited correla-
tion between an analogue’s ability to inhibit transport
of spermidine and its ability to inhibit cell growth in
the presence of DFMO. Some analogues with Ki values
below 100 nM failed to give low EC50 values in combina-
tion with DFMO. Examples of this type of analogue
include 14 and 24. A possible explanation for this
observation is that in addition to competitively inhibit-
ing transport of spermidine, these analogues act as
substrates for the polyamine transporter, thereby enter-
ing and supplying the cells with their necessary
polyamines. The ability of analogues 14, 18, 24, 30, and
31 to rescue the cells from the effect of 230 µM DFMO
in the absence of spermidine was tested (data not
shown). After 6 days in the presence of 230 µM DFMO,
cell growth was inhibited by 60%. 14 and 24 were able
to rescue cells from these growth inhibitory effects of
DFMO. The analogues 18, 30, and 31 did not rescue
cells from these growth inhibitory effects of DFMO.
These results suggested that analogues 14 and 24 were
substrates while analogues 18, 30, and 31 were not
substrates for the polyamine transporter. Alternatively,
if analogues 18, 30, and 31 were substrates for the
transporter, they could not substitute for the growth
functions of the natural polyamines. An additional
possibility is that conjugates 14 and 24 are metabolized
and the resulting products supply the cells with their
required polyamines.
The amino acids containing aromatic groups were
examined next. The L-Trp-Spm (32) (Ki ) 119 nM),
D-Trp-Spm (33) (Ki ) 63 nM), and Tyr-Spm (35) (Ki )
116 nM) conjugates all had good activity, whereas the
Phe-Spm conjugate (34) (Ki ) 443 nM) had diminished
activity.
Cell Gr ow th In h ibition : EC50 in MDA-MB-231
Cells Gr ow n in th e P r esen ce of DF MO a n d Sp er -
m id in e. A 6-day cell growth assay was used to char-
acterize the ability of the amino acid/spermine amides
to work in concert with the ODC inhibitor DFMO in the
presence of added spermidine (0.50 or 1.0 µM) and
aminoguanidine (1 mM) (Figure 1). In this assay, no
growth inhibition was observed with DFMO alone due
to the ability of the cells to meet their polyamine needs
by the uptake of the spermidine added to the culture
medium. If the added analogues prevented the uptake
of the exogenously added spermidine, growth inhibition
due to polyamine depletion was expected. This assay
should identify effective transport inhibitors under
conditions that mimic in vivo conditions.
Given the promising results shown by the use of 31
in combination with DFMO in inhibiting the growth of
MDA-MB-231 cells, we next explored its growth effect
on other tumor cell lines. The results in Table 3 showed
that 31 in combination with DFMO demonstrated a
good cytostatic effect against 9 out of 10 cell lines tested.
For each cell line tested, a DFMO titration curve,
without addition of SPD, was used to determine the
optimal inhibitory concentration of DFMO (0.23-5.0
Analysis of the data from this assay using the novel
polyamine analogues described in Table 2 showed that
several of the conjugates were able to effectively inhibit
the growth of MDA-MB-231 cancer cells in combination
with 230 µM DFMO even in the presence of 1.0 µM
added spermidine. The analogues showing the best
activity in this assay include 31 (Ki ) 32 nM) with an
EC50 of 5 µM and the analogue 30 (Ki ) 47 nM), which

3638 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Burns et al.
Ta ble 3. Growth Inhibition by Polyamine Depletion in
Multiple Tumor Typesa
DFMO
31 + DFMO
EC50 (µM)
concn
(mM)
tumor type
cell line
breast carcinoma
prostate carcinoma
MDA-MB-231
PC-3
DU 145
LNCaP‚FGC
T-24
SK-Mel-5
SK-Mel-28
A375
NCI H157
NCI H226
4.8
5.3
5.0
2.6
1.6
0.23
1.0
1.0
5.0
5.0
5.0
5.0
5.0
0.23
3.0
bladder carcinoma
melanoma
14
3.6
1.5
140
>300
F igu r e 3. ODC activity in MDA cells. MDA-MB-231 cells were
treated for 3 days with SPD (1.0 µM), DFMO (0.50 mM), or
Lys-Spm (100 µM) in the combinations shown. Cells were
harvested for ODC activity assay while in log growth. The
activity was normalized to protein concentration. All experi-
ments contained 1.0 mM AG.
lung carcinoma
(nonsmall cell)
a
31 EC50 values were determined in a 6-day growth assay with
the optimal DFMO concentration for each cell line. All assays
performed in the presence of 1.0 mM AG and 1.0 µM spermidine.
31 to work in combination with DFMO to significantly
reduce intracellular putrescine and spermidine levels
under conditions that mimic the in vivo tumor environ-
ment.
ODC activity was measured in MDA-MB-231 cells
following treatment with various drugs (Figure 3). It is
theorized that due to the regulated control of ODC
activity in the cells the inclusion of transportable
spermidine in the culture medium caused a lowering of
the measured ODC activity to 12% of the control value
seen with no treatment. Treatment with both spermi-
dine and DFMO gave, as expected, a similar lowered
value of ODC activity in comparison with control values.
When the cells were treated with spermidine and 31,
the ability of the polyamine transport inhibitor to
prevent the uptake of spermidine caused the ODC
activity of the cells to rise to slightly higher than control
levels (149%). This result may explain the observed
increased levels of putrescine following 31 treatment
(Figure 2). This result shows that treatment with 31
does not inhibit ODC activity of the cells. It also
supports the conclusion that 31 prevents the uptake of
endogenously supplied spermidine. Finally, Figure 3
shows that treatment with a combination of spermidine,
DFMO, and 31 gave the expected result of lowered ODC
activity.
F igu r e 2. Polyamine levels in MDA-MB-231 cells after 6 days
with 31, DFMO, or the combination of both (all with 1.0 µM
spermidine). MDA-MB-231 cells were grown for 6 days (1
week) with 500 µM DFMO or 60 µM 31 or both. All flasks
received 1.0 mM AG and 1.0 µM SPD. Cells were counted,
washed, lysed in perchloric acid, and dansylated and polyamine
levels determined by HPLC. Each point is the mean of three
experiments. The bars represent standard deviations.
mM). Again, 1.0 µM SPD was included to ensure that
growth inhibition reflected inhibition of transport. Most
EC50 values were in the low micromolar range (Table
3). The exceptions were the two nonsmall cell lung
carcinoma cell lines. Their growth responses may reflect
cell-line- or tissue-specific differences in transport or
metabolism.
Discu ssion
P olya m in e Dep let ion : Ab ilit y of Com b in a t ion
Th er a p y To Dep lete In tr a cellu la r P olya m in es. The
intracellular polyamine levels in MDA-MB-231 cells
were measured following a 6-day drug treatment period.
The polyamine levels were measured by HPLC using
fluorescent detection following precolumn derivativation
with dansyl chloride.41 Figure 2 shows the results after
various drug treatments. Following a 6-day incubation
with DFMO alone (500 µM), 1 and 2 levels were not
significantly reduced in MDA-MB-231 cells due to the
inclusion of adequate transportable spermidine in the
culture medium. Polyamine levels after treatment with
60 µM 31 together with 1.0 µM spermidine raised the
levels of 1 in the cells but only had minimal effects on
2 and 3 levels. In contrast, a significant reduction of
the intracellular levels of putrescine and spermidine
was observed when 60 µM 31 and 500 µM DFMO was
used in combination in the presence of 1.0 µM spermi-
dine. These results clearly demonstrate the ability of
The genes for the polyamine transporter subunits
have been cloned from Escherichia coli,42 and one was
recently identified in yeast.43 The genes for the mam-
malian transporter await identification. For this reason
it is difficult to determine whether different transporters
exist for each polyamine. The PotD subunit of the E.
coli transporter has been crystallized, and its X-ray
structure has been determined.44 Several researchers
have studied the ability of polyamine analogues to
inhibit the uptake of [3H]spermidine into cells. Bergeron
and co-workers studied the effect of the addition of
different alkyl group substitutions on the terminal
nitrogen atoms of spermidine or spermine analogues.45
They showed that larger alkyl groups diminished the
ability to prevent uptake of radiolabeled spermidine.
They later concluded that increases in the number of
methylenes between the nitrogen atoms decreased the
ability to compete for [3H]spermidine uptake.46 Of
greater importance to the present work was their

Amino Acid/ Spermine Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3639
Ta ble 4. Polyamine Km Values and Inhibition Constants (Ki
Values) with 31 on MDA-MB-231 Cellsa
conclusion that the polyamine transport apparatus
requires only three cationic centers for polyamine
recognition and transport.47
Ki(31)
substrate
(nM + SD)
characterization
competitive
Several research groups have reported the search for
compounds that would prevent the uptake of extracel-
lular polyamines and thus increase the clinical utility
of polyamine biosynthesis inhibitors. Poulin and co-
workers synthesized 2,2′-dithiobis(N-ethylspermine-5-
carboxamide) (DESC) and characterized its inhibition
of polyamine uptake into ZR-75-1 cells. Ki values of 1.6
( 5.0, 2.7 ( 1.1, and 5.0 ( 7.0 µM against putrescine,
spermidine, and spermine, respectively, were reported.48
Despite the advantage of it not being a transport
substrate, the addition of 0.3 µM spermidine fully
restored growth to cells treated with the combination
of 1.0 mM DFMO and 50 µM DESC. Analysis of the
levels of polyamine in the cells showed that spermidine
accumulation was not prevented by large concentrations
of this compound. Furthermore, significant cytotoxicity
was observed when 200 µM DESC was used alone.
Poulin has subsequently published results on a new
series of polyamine transport inhibitors.49 This series
of compounds was described as dimers of spermidine
or norspermidine and have six ionizable amino func-
tions. Several of these analogues have activities as
polyamine transport inhibitors in T-47D human breast
cancer cells that are much better than the corresponding
apparent affinities of putrescine or spermidine. The best
analogues described have affinities nearly equal to that
of spermine. Minchin and co-workers described a series
of polypyridinium quaternary salts as putrescine uptake
inhibitors in B16 melanoma cells.50 Despite having Ki
values in the low micromolar range, combination with
DFMO had no effect on the growth inhibitory properties
of the described compounds. Again, significant cytotox-
icity was observed when the compounds were tested
alone. Finally, a macromolecular polymer (∼25 kDa) of
spermine and glutaraldehyde was shown to inhibit
polyamine uptake in the micromolar range, but its
cytotoxicity again limits its usefulness.51 Two groups
have analyzed literature examples of numerous poly-
amine analogues’ ability to inhibit [3H]spermidine up-
take into L1210 cells by CoMFA52 and QSAR53 methods.
A recent report explored the effects of acylation of
various nitrogen atoms of spermidine or spermine on
the inhibition of polyamine uptake together with the
resulting effects on polyamine pools in E. coli cells.54
[3H]putrescine
24 ( 1
n ) 7
[3H]spermidine
32 ( 2
n ) 4
competitive
competitive
[
14C]spermine
84 ( 2
n ) 3
a
Multiple concentrations of substrate and 31 were used to
determine Ki values in 15-min transport assays.
ral product 6, had moderate ability to inhibit the
transport of [3H]spermidine in MDA-MB-231 cells, we
initiated the synthesis of a series of simple amino acid/
spermine amide conjugates (Table 2). These have at
least three cationic centers in their polyamine portion
and thus satisfy Bergeron’s criteria for recognition by
the polyamine transporter.47
The novel series of amino acid/spermine conjugates
described in the present paper included compounds with
significant activity as inhibitors of polyamine uptake
into MDA-MB-231 cells. A comparison of their Ki values,
derived from competition with spermidine, gives a
ranking of their potency. It was interesting to note that
minor variations in an analogue’s structure gave great
changes in its relative potency as a transport inhibitor
(compare 7 with 10, 2-(1-Adam)-Spm (11) with 1-Adam-
Spm (12), 14 with 16, or 22 with 23). Furthermore,
changing the stereochemistry of the R-carbon atom of
the amino acid portion of the molecule from the L- to
the D-configuration showed the possibility of slight
increases in activity (compare 18 with 19 and 32 with
33). On the basis of this analysis and further biological
testing discussed below, 31 was selected as a lead
compound for extensive biological testing including in
vivo efficacy in combination with DFMO.59,60 Measure-
ment of the Ki values for 31 versus putrescine, spermi-
dine, and spermine was performed. As shown in Table
4, this molecule showed potent competitive inhibition
against each polyamine. Furthermore, this molecule’s
activity as a transport inhibitor in MDA-MB-231 cells
exceeded the apparent affinities of the natural poly-
amines putrescine, spermidine, and spermine several-
fold.
Satisfying one of the major goals of the present
research, all of the novel analogues described here had
EC50 values greater than 300 µM when tested alone in
the 6-day growth assay of MDA-MB-231 breast cancer
cells. It was only when an effective polyamine transport
inhibitor, such as 31, was used in combination with the
polyamine biosynthesis inhibitor DFMO that growth
inhibitory effects were observed. As shown in Figure 1,
this effect was seen even in the presence of 0.50 µM
extracellular spermidine added to the cell culture
medium. This amount of spermidine, when added
together with DFMO alone, completely abolished the
growth inhibitory effects of this polyamine biosynthesis
inhibitor (data not shown). The growth inhibition seen
with dual-compound therapy was seen in a variety of
cell lines representing multiple tumor types (Table 3).
An additional requirement placed on any molecule
that would function as a polyamine transport inhibitor
in a chemotherapeutic setting would be its lack of
polyamine-replacing or -mimicking ability. A test of the
A great number of polyamine amide natural products
have recently been discovered in the venom of arthro-
pods (spider and wasp).55 These acylpolyamine ana-
logues have been shown to have specific and strong
interactions with the neuromuscular junctions of in-
sects.56 With this capability, these toxins give the insect
predators the ability to paralyze or kill their prey. Most
of these natural products have the common molecular
features of a polyamine moiety (many with structurally
diverse polyamine analogues) connected through an
amide with an aromatic amino acid structural analogue.
Simpler synthetic analogues have been sought that
attempt to maximize interactions with either crustacean
neuromuscular synapses38 or mammalian glutamate
receptors.57,58 Given our discovery that the polyamine
amide spider toxin 4 and its synthetic analogue 5,
together with the immunosuppressant polyamine natu-

3640 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Burns et al.
(1-Naphthylacetyl)spermine was purchased from RBI. 15-
Deoxyspergualin was a generous gift from Dr. Paul Gladstone.
ability of a polyamine analogue’s ability to act as a
polyamine surrogate, or to be metabolized to the natural
polyamines themselves, was its ability to overcome the
growth inhibitory effects of DFMO, in the absence of
added spermidine. The implication of this test is that
either the analogue does not act as a substrate for the
polyamine uptake system or it does not function as a
polyamine once it enters the cell. With the inability of
<300 µM 31 to rescue growth from the effects of DFMO
treatment, our working assumption is that it does not
enter the cell. Experiments are now in progress to
determine whether 31 accumulates in cells.
The final, and ultimately the most important, crite-
rion placed on our search for a polyamine transport
inhibitor was its ability to deplete intracellular poly-
amines when used in combination with a biosynthesis
inhibitor. The data shown in Figure 2 confirm our
design goals by showing that, even in the presence of
sufficient spermidine in the culture medium, the com-
bination of 31 and DFMO greatly reduces the intracel-
lular levels of putrescine and spermidine in MDA-MB-
231 cells. This reduction in putrescine and spermidine
levels is reflected by the reduction in the growth of the
MDA-MB-231 cells (60% of control). Even after 2 weeks
of growth arrest, the cells will regrow upon removal of
the drug (data not shown). Therefore, this is a cytostatic
treatment. It is unclear why the spermine levels remain
near their control levels. It has been suggested in the
literature that the intracellular pool of spermine is
completely bound to cellular anionic components.3 Pre-
liminary in vivo testing showed no gross acute toxicities
for 31 that might be expected on the basis of its
structural relationship to the acylpolyamine spider
toxins.61 We speculate that the lack of an aromatic
moiety common to most, if not all, of the spider toxins
limits the neurotoxicity of 31.
Syn th etic Meth od s. Gen er a l In for m a tion . All chemical
reagents and starting materials were of the highest grade
available and were used without further purification. Thin-
layer chromatography analysis of crude reaction products and
column chromatography were performed using Merck F254
silica gel plates and Baker 40 µm flash chromatography
packing, respectively. TLC analysis used the following solvent
system with detection by ninhydrin staining: 2-propanol/
pyridine/glacial acetic acid/H2O, 4:1:1:2. The Rf values were
0.1 for spermine and generally 0.5 for the mono- and 0.7 for
the disubstituted products. 1H and 13C NMR spectra were
recorded at 200, 300, or 500 MHz and 75.5 or 125.8 MHz,
respectively, on a Bruker AC200, AF300, or WM500 spectrom-
eter at the University of Washington, Seattle. Chemical shifts
are relative to external 3-(trimethylsilyl)-1-propanesulfonic
acid, sodium salt. HRMS was run at The Scripps Research
Institute using MALDI FTMS with DHB matrix on an IonSpec
HiRes MALDI instrument.
Syn t h esis of Am in o Acid /Sp er m in e Con ju ga t es. The
following two procedures are representative of the synthesis
of the conjugates. Specific details about each conjugate are
included with the spectroscopic characterizations below.
Meth od A: N1-Sp er m in e L-ter t-Bu tylglycin yl Am id e
(20). 4-Nitr op h en yl N-tBoc-L-ter t-bu tylglycin a te. A het-
erogeneous solution of 0.463 g (2 mmol) of N-tBoc-tert-butylg-
lycine (from Novabiochem), 0.334 g (1.2 equiv) of 4-nitrophenol,
and 0.495 g (1.2 equiv) of 1,3-dicyclohexylcarbodiimide in 20
mL of dry EtOAc was stirred for 16 h at ambient temperature
under argon. The solids were filtered over a pad of Celite, and
the pad was washed twice with additional portions of EtOAc.
The combined filtrates were evaporated, and the resulting solid
was purified by column chromatography over silica gel using
95:5 CHCl3/EtOAc to yield 0.60 g (85%) of clear oil.
N1-Sp er m in e N-tBoc-L-ter t-bu tylglycin yl Am id e. To the
clear, homogeneous solution of 136 mg (1.2 equiv) of spermine
(free base form) in 15 mL of MeOH was added dropwise
through a funnel a solution of 198 mg (0.56 mmol) of 4-nitro-
phenyl N-tBoc-tert-butylglycinate in 15 mL of MeOH at ambi-
ent temperature. After 2 h, the resulting yellow-green solution
was checked by the above TLC method, which showed the
expected mixture of di-, mono-, and unsubstituted products
had been formed. The solvents were evaporated, and the crude
product was suspended in 25 mL of H2O. Upon adjusting the
pH to 4-5 by the addition of 1 N HCl, a homogeneous, clear
solution was produced. This was loaded onto a 1 × 30 cm
Con clu sion s
In this paper we describe the synthesis and charac-
terization of a series of simple spermine/amino acid
conjugates, some of which potently inhibit the uptake
of spermidine into MDA-MB-231 breast cancer cells. The
presence of an amide in the functionalized polyamine
appeared to add to the affinity for the polyamine
transporter. The extensive biological characterization
of an especially potent analogue from this series, 31,
showed this molecule will be an extremely useful tool
for use in polyamine research. It was shown that the
use of 31 in combination with DFMO led to a cytostatic
growth inhibition of a variety of cancer cells, even when
used in the presence of an extracellular source of
polyamines. It was shown that this combination ef-
fectively reduced the cellular levels of putrescine and
spermidine while not affecting the levels of spermine.
Despite the structural similarity between 31 and the
acylpolyamine spider toxin natural products, prelimi-
nary in vivo data suggest that this compound does not
show neurotoxicity to animals at dose levels necessary
for transport inhibition.59,60 These facts together with
the nontoxic nature of 31 make it an ideal lead for
further anticancer development.
+
column of BioRex 70 resin that was in the NH4 salt form.
The column was washed with 50 mL of H2O followed by an
800 mL gradient of 0-2 N NH4OH to elute the products into
80 fractions of 10 mL each. These fractions were analyzed by
the above TLC system, and the desired monosubstituted
product containing fractions were combined and evaporated
to give 105 mg (45%) of the product in its free base form.
N1-Sp er m in e L-ter t-Bu tylglycin yl Am id e (20). A 6.0 mL
portion of 6 N HCl was added to the clear solution of 98 mg
(0.24 mmoL) of N1-spermine N-tBoc-L-tert-butylglycinyl amide
in 6.0 mL of MeOH. The resulting homogeneous colorless
solution was stirred at ambient temperature for 3 h, after
which the solvent was evaporated to give 110 mg (99%) of
desired 20 as its tetrahydrochloride salt. 1H NMR (D2O, δ):
3.50 (s, 1H), 3.15 (t, 2H), 2.91 (m, 12H), 1.90 (m, 2H), 1.74 (m,
2H). 1.58 (m, 4H), 0.85 (s, 9H). 13C NMR (D2O, ppm): 171.2,
67.9, 64.4, 49.6, 49.5, 47.8, 47.1, 39.2, 39.0, 35.3, 28.2, 28.0,
26.3, 25.3. HRMS: m/z calcd for C16H37N5O (M + H) 316.3076,
found 316.3063.
Meth od B: N1-Sp er m in e L-Lysin yl Am id e (31).
N1-Sp er m in e
Nr,NE-Bis(ca r b ob en zyloxy)-L-lysin yl
Am id e. A clear solution of 1.02 g (2.0 mmol) of NR,Nꢀ-bis-
(carbobenzyloxy)-L-lysine N-hydroxysuccinimide ester (Sigma,
St. Louis, MO) in 50 mL of methylene chloride was added
dropwise over 15 min at room temperature to a clear, homo-
geneous solution of 0.486 g (2.4 mmol, 1.2 equiv) of spermine
in 50 mL of methylene chloride. A white precipitate formed.
Exp er im en ta l Section
The J oro spider toxin J STx-3 was purchased from Calbio-
chem.

Amino Acid/ Spermine Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3641
After the resulting solution was stirred for 4 h, TLC analysis
showed the expected mixture of di-, mono-, and nonacylated
spermine derivatives had formed. The solvent was evaporated
to give a white solid. This crude product was suspended in
3:1 H2O/MeOH (25 mL), and the pH was adjusted to 4-5 using
1 N HCl. The resulting clear, homogeneous solution was
(m, 2H), 1.78 (m, 4H). 13C NMR (D2O, ppm): 175.0, 49.6, 49.5,
47.8, 47.1, 39.2, 38.8, 38.3, 34.5, 28.1, 26.3, 25.4 (2C). HRMS:
m/z calcd for C13H31N5O (M + H) 274.2607, found 274.2604.
t
N1-Sp er m in e L-Ala n yl Am id e (16). 4-Nitrophenyl Boc-
L-alanine ester was obtained from Novabiochem and coupled
to spermine in 56% yield. The tBoc protecting group was
removed through acid treatment as in method A to give 16 as
its tetrahydrochloride salt in 98% yield. 1H NMR (D2O, δ): 4.08
(quartet, 1H), 3.19 (t, 2H), 3.08 (m, 10H), 2.13 (m, 2H), 1.96
(m, 2H), 1.79 (m, 4H), 1.52 (d, 3H). 13C NMR (D2O, ppm):
173.6, 51.7, 49.6, 49.5, 47.8, 47.2, 39.2, 40.0, 28.1, 26.4, 25.4
(2C), 19.2. HRMS: m/z calcd for C13H31N5O (M + H) 274.2607,
found 274.2604.
+
applied to a 1 × 30 cm column of BioRex 70 resin in its NH4
form. The column was washed with 50 mL of 4:1 H2O/MeOH,
and then a 800 mL gradient of 0-2 N NH4OH containing 25%
MeOH was used to elute the products into 80 fractions of 10
mL each. These fractions were analyzed by the above TLC
system, and the desired monosubstituted product containing
fractions were combined and evaporated to give 527 mg (44%)
of the product as its free base form.
N1-Sp er m in e r-Am in oisobu tyr yl Am id e (17). N-tBoc-R-
aminoisobutyric acid was obtained from Sigma and was
converted to its 4-nitrophenyl active ester as in method A
above. Coupling to spermine gave 57% of the protected
conjugate as its free base. Deprotection via acid treatment gave
N1-Sp er m in e L-Lysin yl Am id e (31). A mixture of 500 mg
(0.83 mmol) of N1-spermine NR,Nꢀ-bis(carbobenzyloxy)-L-lysinyl
amide and 300 mg of Pd(OH)2 in 50 mL of 1:1 EtOH/H2O
containing 10 mL of 2 N HCl was stirred under an atmosphere
of H2 gas using a balloon reservoir. After 18 h at ambient
temperature the catalyst was filtered off over a pad of Celite,
and the pad was washed twice each with EtOH, H2O, and then
1 N HCl. The combined filtrates were evaporated to give 420
mg (98%) of product 31 as its pentahydrochloride salt. 1H NMR
(D2O, δ): 4.09 (t, 1H), 3.39 (m, 2H), 3.17 (m, 14H), 2.16 (m,
2H), 1.99 (m, 4H), 1.85 (m, 4H), 1.51 (m, 2H). 13C NMR (D2O,
ppm): 173.2, 56.4, 50.3, 50.2, 48.5, 47.9, 42.4, 40.0, 39.8, 33.6,
29.6, 28.8, 27.1, 26.4 (2C), 24.7. HRMS: m/z calcd for C16H38N6O
(M + H) 331.3185, found 331.3173.
1
17 as its tetrahydrochloride salt in 92% yield. H NMR (D2O,
δ): 3.37 (t, 2H), 3.14 (m, 10H), 2.13 (m, 2H), 1.96 (m, 2H),
1.82 (m, 4H), 1.64 (s, 6H). 13C NMR (D2O, ppm): 173.5, 57.6,
47.7, 47.6, 45.9, 45.3, 37.3 (2C), 26.2, 24.4, 24.0 (2C), 23.5 (2C).
HRMS: m/z calcd for C14H33N5O (M + H) 288.2763, found
288.2762.
N1-Sp er m in e L-Va lin yl Am id e (18). N-tBoc-L-valine N-
hydroxysuccinimide ester was obtained from NovaBiochem
and coupled to spermine using method B (48% yield as free
base). Deprotection by acid treatment gave 18 as its tetrahy-
N1-Sp er m in e 2-(1-Ad a m a n tyl)a ceta m id e (11). The 4-ni-
trophenyl ester of 2-(1-adamantyl)acetic acid was produced
(52% yield) and coupled to spermine via method A described
for 20 above. Purification over BioRex 70 resin gave pure 11
as its free base (17% yield). It was converted to its trihydro-
chloride salt for analysis and biological testing. 1H NMR (D2O,
δ): 2.98 (t, 2H), 2.82 (m, 10H), 1.80 (m, 2H), 1.76 (s, 3H), 1.63
(m, 6H), 1.48 (m, 4H), 1.36 (m, 4H), 1.22 (s, 6H). HRMS: m/z
calcd for C22H42N4O (M + H) 379.3437, found 379.3443.
1
drochloride salt in 99% yield. H NMR (D2O, δ): 3.36 (d, 1H),
3.18 (m, 2H), 3.11 (m, 10H), 2.02 (m, 3H), 1.82 (m, 2H), 1.54
(m, 4H), 0.90 (d, 6H). 13C NMR (D2O, ppm): 172.1, 61.3, 49.6,
49.5, 47.9, 47.2, 39.2, 39.1, 32.4, 28.1, 26.4, 25.4 (2C), 20.3,
19.7. MS (ESI+): 302 (M + H). HRMS: m/z calcd for C15H35N5O
(M + H) 302.2920, found 302.2914.
N1-Sp er m in e D-Va lin yl Am id e (19). The 4-nitrophenyl
ester of N-tBoc-D-valine (from Sigma) was synthesized via
method A (66% yield). Coupling to spermine and purification
N1-Sp er m in e 1-Ad a m a n tyl Am id e (12). A CH2Cl2 solu-
tion of spermine and triethylamine was treated with a CH2-
Cl2 solution of 1-adamantanecarbonyl chloride. After 2 h, the
solvents were evaporated and the residue was purified via
gave 48% of the Boc-protected conjugate. Deprotection using
t
acid gave 19 as its tetrahydrochloride salt in 99% yield. 1H
NMR and 13C NMR were the same as for 18.
N1-Sp er m in e L-Isoleu cin yl Am id e (21). N-tBoc-L-isoleu-
cine 4-nitrophenyl ester was obtained from Sigma and coupled
to spermine via method A (60% yield). Deprotection by acid
treatment gave 21 as its tetrahydrochloride salt in 99% yield.
1H NMR (D2O, δ): 3.84 (d, 1H), 3.37 (t, 2H), 3.11 (m, 10H),
2.09 (m, 2H), 1.96 (m, 3H), 1.78 (m, 4 H), 1.50 (m, 1H), 1.26
(m, 1H), 0.99 (d, 3H), 0.93 (t, 3H). 13C NMR (D2O, ppm): 172.1,
60.4, 49.5, 47.8, 47.1, 39.2, 39.0, 38.8, 28.0, 26.8, 26.3, 25.4
(2C), 25.2, 16.7, 13.1. HRMS: m/z calcd for C16H37N5O (M +
H) 316.3076, found 316.3064.
+
method A above using BioRex 70 (NH4 form) resin. Pure 12
in its free base form was obtained (19% yield) and converted
to its trihydrochloride salt for analysis and biological testing.
1H NMR (D2O, δ): 3.24 (t, 2H), 3.00 (m, 10H), 1.95 (m, 4H),
1.74 (m, 10H), 1.62 (m, 9H). HRMS: m/z calcd for C21H40N4O
(M + Na) 387.3100, found 387.3085.
N1-Sp er m in e 2-(2-In d olyl)a ceta m id e (13). The 4-nitro-
phenyl ester of 2-(2-indolyl)acetic acid was produced (77%
yield) and coupled to spermine via method A described for 20
above. Purification over BioRex 70 resin gave pure 13 as its
free base (48% yield). It was converted to its trihydrochloride
salt for analysis and biological testing. 1H NMR (D2O, δ): 7.43
(d, 1H), 7.38 (d, 1H), 7.17 (s, 1H), 7.11 (t, 1H), 7.02 (t, 1H),
3.56 (s, 2H), 3.08 (t, 2H), 1.93 (m, 4H), 2.84 (t, 2H), 2.61 (m,
4H), 1.94 (m, 2H), 1.62 (m, 2H), 1.48 (m, 4H). 13C NMR (D2O,
ppm): 178.3, 138.7, 129.0, 127.6, 124.6, 122.1, 120.8, 114.6,
110.1, 49.4, 49.2, 47.3, 47.0, 39.1, 38.4, 34.9, 28.0, 26.3, 25.2,
25.0. HRMS: m/z calcd for C20H33N5O (M + H) 360.2763, found
360.2773.
N1-Sp er m in e L-Leu cin yl Am id e (22). N-tBoc-L-leucine
4-nitrophenyl ester was obtained from Sigma and coupled to
spermine via method A (40% yield). Deprotection by acid
treatment gave 22 as its tetrahydrochloride salt in 99% yield.
1H NMR (D2O, δ): 3.98 (t, 1H), 3.33 (m, 2H), 3.09 (m, 10 H),
2.09 (m, 2H), 1.91 (m, 2H), 1.74 (m, 7H), 0.93 (dd, 6H). 13C
NMR (D2O, ppm): 173.2, 54.6, 49.6, 47.8, 47.2, 42.4, 39.2, 39.0,
28.0, 27.9, 26.5, 26.3, 25.4 (2C), 24.4, 23.8. HRMS: m/z calcd
for C16H37N5O (M + H) 316.3076, found 316.3073.
N1-Sp er m in e L-Cycloh exyla la n yl Am id e (23). The 4-ni-
trophenyl ester of N-tBoc-L-cyclohexylalanine (from Sigma) was
synthesized via method A (82% yield). Coupling to spermine
and purification gave 59% of the tBoc-protected conjugate.
Deprotection using acid gave 23 as its tetrahydrochloride salt
in 98% yield. 1H NMR (D2O, δ): 4.03 (t, 1H), 3.36 (m, 2H),
3.12 (m, 10H), 2.11 (m, 2H), 1.95 (m, 2H), 1.77 (m, 12H), 1.11
(m, 5H). 13C NMR (D2O, ppm): 173.4, 54.0, 49.6, 49.5, 47.8,
47.1, 41.0, 39.2, 39.0, 35.7, 35.2, 34.6, 28.3, 28.2, 28.0, 26.3,
25.4. HRMS: m/z calcd for C19H41N5O (M + H) 356.3389, found
356.3375.
N1-Sp er m in e Glycin yl Am id e (14). 4-Nitrophenyl N-tBoc-
glycinate was obtained from Novabiochem and directly coupled
to spermine in 43% yield using method A above. Deprotection
in 3 N HCl gave the desired 14 as its tetrahydrochloride salt
in 93% yield. 1H NMR (D2O, δ): 3.91 (s, 2H), 3.43 (t, 2H), 3.20
(m, 10H), 2.21 (m, 2H), 2.18 (m, 2H), 1.87 (m, 4H). 13C NMR
(D2O, ppm): 175.4, 47.8, 47.7, 46.0, 45.3, 41.2, 37.4, 37.1, 26.2,
24.5, 23.6 (2C). HRMS: m/z calcd for C12H29N5O (M + H)
260.2450, found 260.2442.
t
N1-Sp er m in e â-Ala n yl Am id e (15). 4-Nitrophenyl Boc-
â-alanine ester was obtained from Sigma and coupled to
spermine in 39% yield via method A above. Deprotection gave
98% of 15 as its tetrahydrochloride salt. 1H NMR (D2O, δ):
3.29 (m, 4H), 3.10 (m, 10H), 2.68 (t, 2H), 2.09 (m, 2H), 1.90
N1-Sp er m in e L-P r olin yl Am id e (24). N-tBoc-L-proline
N-hydroxysuccinimide ester was obtained from Sigma and
coupled to spermine using method B (56% yield as free base).
Deprotection by acid treatment gave 24 as its tetrahydrochlo-

3642 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Burns et al.
ride salt in 94% yield. 1H NMR (D2O, δ): 4.45 (t, 1H), 3.53 (m,
2H), 3.50 (t, 2H), 3.18 (m, 14H), 2.19 (m, 4H), 2.01 (m, 2H),
1.86 (m, 2H). 13C NMR (D2O, ppm): 172.9, 63.2, 57.1, 50.4,
50.3, 49.8, 48.6, 47.9, 40.1, 40.0, 33.1, 28.8, 27.3, 27.2, 26.3.
HRMS: m/z calcd for C15H33N5O (M + H) 300.2763, found
300.2762.
1.59 (m, 2H). 13C NMR (D2O, ppm): 170.5, 136.6, 127.3, 126.0,
123.0, 120.2, 118.6, 112.7, 108.8, 54.6, 47.7, 47.4, 45.6, 45.5,
37.2, 37.1, 27.7, 25.4, 24.6, 23.9 (2C). MS (ESI+): 389 (M +
H). HRMS: m/z calcd for C21H36N6O (M + Na) 411.2848, found
411.2835.
N1-Sp er m in e D-Tr yp top h a n yl Am id e (33). The 4-nitro-
phenyl ester of N-tBoc-D-tryptophan (from Sigma) was syn-
thesized via method A (85% yield). Coupling to spermine and
N1-Sp er m in e L-Asp a r a gin yl Am id e (25). N-tBoc-L-aspar-
agine 4-nitrophenyl ester was obtained from Sigma and
coupled to spermine via method A (44% yield). Deprotection
by acid treatment gave 25 as its tetrahydrochloride salt in 99%
t
purification gave 54% of the Boc-protected conjugate. Depro-
tection using acid gave 33 as its tetrahydrochloride salt in 94%
1
1
yield. H NMR (D2O, δ): 4.33 (m, 1H), 3.30 (m, 2H), 3.10 (m,
yield. H NMR and 13C NMR were the same as for 32.
12H), 2.09 (m, 2H), 1.92 (m, 2H), 1.78 (m, 4H). 13C NMR (D2O,
ppm): 173.8, 173.7, 52.3, 52.1, 49.6, 47.7, 47.1, 39.1, 38.7, 37.4,
37.0, 28.1, 26.3, 25.4. HRMS: m/z calcd for C14H32N6O2 (M +
H) 317.2665, found 317.2662.
N1-Sp er m in e L-P h en yla la n yl Am id e (34). N-tBoc-L-phe-
nylalanine 4-nitrophenyl ester was obtained from Sigma and
coupled to spermine via method A (52% yield). Deprotection
by acid treatment gave 34 as its tetrahydrochloride salt in 97%
N1-Sper m in e L-Glu tam in yl Am ide (26). N-tBoc-l-glutamine
4-nitrophenyl ester was obtained from Sigma and coupled to
spermine via method A (57% yield). Deprotection by acid
treatment gave 26 as its tetrahydrochloride salt in 99% yield.
1H NMR (D2O, δ): 4.05 (m, 1H), 3.34 (t, 2H), 3.13 (m, 10H),
2.48 (m, 2H), 2.18 (m, 4H), 1.93 (m, 2H), 1.79 (m, 4H). 13C NMR
(D2O, ppm): 176.1, 170.1, 57.7, 53.2, 47.7, 45.8, 45.2, 37.3, 36.8,
29.9, 26.3, 26.0, 24.4, 23.5 (2C). HRMS: m/z calcd for
yield. H NMR (D2O, δ): 7.46 (m, 5H), 4.28 (dd, 1H), 3.25 (m,
1
12H), 2.85 (t, 2H), 2.19 (m, 2H), 1.86 (m, 6H). 13C NMR (D2O,
ppm): 171.7, 136.7, 132.1, 131.8, 130.6, 83.8, 57.2, 49.7, 49.6,
47.7, 47.2, 39.5, 39.3, 39.0, 27.7, 26.4, 25.4. HRMS: m/z calcd
for C19H35N5O (M + H) 350.2920, found 350.2904.
N1-Sp er m in e L-Tyr osin yl Am id e (35). N-tBoc-O-benzyl-
L-tyrosine N-hydroxysuccinimide ester was obtained from
Novabiochem and coupled to spermine using method B (50%
yield as free base). The O-benzyl protecting group was removed
via the hydrogenation procedure in method B. Deprotection
by acid treatment gave 35 as its tetrahydrochloride salt in 90%
yield. 1H NMR (D2O, δ): 7.20 (d, 2H), 6.96 (d, 2H), 4.15 (t,
1H), 3.36 (m, 2H), 3.18 (m, 10H), 2.77 (t, 2H), 2.14 (m, 2H),
1.82 (m, 6H). HRMS: m/z calcd for C19H35N5O2 (M + H)
366.2869, found 366.2852.
C
15H34N6O2 (M + H) 331.2821, found 331.2826.
N1-Sp er m in e L-Meth ion yl Am id e (27). N-tBoc-L-methion-
ine 4-nitrophenyl ester was obtained from Sigma and coupled
to spermine via method A (45% yield). Deprotection by acid
treatment gave 22 as its tetrahydrochloride salt in 92% yield.
1H NMR (D2O, δ): 4.10 (t, 1H), 3.34 (m, 2H), 3.10 (m, 12H),
2.60 (t, 2H), 2.18 (m, 2H), 2.11 (s, 3H), 2.07 (m, 2H), 1.78 (m,
4H). 13C NMR (D2O, ppm): 172.1, 55.0, 49.6, 49.5, 47.8, 47.1,
39.1, 32.4, 30.8, 28.0, 26.3, 25.4 (2C), 16.6. MS (ESI+): 334
(M + H). HRMS: m/z calcd for C15H35N5OS (M + H) 334.2640,
found 334.2643.
N1-Sp er m in e L-Ser in yl Am id e (28). N-tBoc-O-benzyl-L-
serine N-hydroxysuccinimide ester was obtained from Sigma
and coupled to spermine using method B (48% yield as free
base). The O-benzyl protecting group was removed via the
hydrogenation procedure in method B. Deprotection by acid
treatment gave 28 as its tetrahydrochloride salt in 94% yield.
1H NMR (D2O, δ): 4.13 (t, 1H), 4.00 (m, 2H), 3.36 (t, 2H), 3.12
(m, 10H), 2.13 (m, 2H), 2.09 (m, 2H), 1.93 (m, 4H). 13C NMR
(D2O, ppm): 168.7, 61.0, 55.4, 47.9, 47.8, 46.0, 45.4, 37.5, 37.4,
36.4, 24.6, 23.7 (2C). HRMS: m/z calcd for C13H31N5O2 (M +
H) 290.2556, found 290.2547.
N1-Sp er m in e L-Th r eon in yl Am id e (29). N-tBoc-O-benzyl-
L-threonine N-hydroxysuccinimide ester was obtained from
Sigma and coupled to spermine using method B (56% yield as
free base). The O-benzyl protecting group was removed via the
hydrogenation procedure in method B. Deprotection by acid
treatment gave 28 as its tetrahydrochloride salt in 90% yield.
1H NMR (D2O, δ): 4.04 (m, 1H), 3.72 (d, 1H), 3.23 (t, 2H), 2.97
(m, 12H), 1.96 (m, 2H), 1.81 (m, 2H), 1.65 (m, 4H), 1.16 (d,
3H). 13C NMR (D2O, ppm): 169.8, 67.2, 58.4, 57.7, 47.4, 46.3,
45.4, 37.6, 37.5, 37.1, 25.3, 24.2 (2C), 20.1. HRMS: m/z calcd
for C14H33N5O2 (M + H) 304.2712, found 304.2715.
Biologica l Meth od s. Cell Cu ltu r e a n d Rea gen ts. The
human carcinoma cell lines were obtained from ATCC (Rock-
ville, MD) and cultured in the recommended medium, serum,
and CO2 concentration. The medium was obtained from
Mediatech, Inc. (Herndon, VA) and the serum from Gibco BRL
(Gaithersburg, MD). Penicillin (50 U/mL), streptomycin (50
µg/mL), and l-glutamine (2 mM) (all from BioWhittaker,
Walkersville, MD) were included in all cultures. DFMO was
obtained from Marion Merrell Dow (Cinncinati, OH). When
cells were cultured with polyamines or any of the novel
analogues, 1 mM aminoguanidine (AG; Sigma) was included
to inhibit serum amine oxidase activity.
P olya m in e Tr a n sp or t a n d Ki Assa ys. To MDA-MB-231
cells in log growth in 24-well plates was added [2,3-3H2]PUT
(DuPont NEN), [2,9-3H2]SPD (DuPont NEN, Boston, MA), or
[1,4-14C2]SPM (Amersham, Arlington Heights, IL) alone or
simultaneously with the polyamine analogue. The cells were
incubated at 37 °C for 15 min, which had previously been
shown to be within the linear range for polyamine uptake. The
cells were then washed three times with PBS and lysed with
0.1% SDS, and the amount of radiolabeled polyamine incor-
poration into the cells was determined by scintillation counting
of the cell lysates. To determine a Ki, four polyamine substrate
concentrations and five inhibitor concentrations together with
a control were tested. The Ki and Km values were calculated
using a Lineweaver-Burke analysis.
N1-Sp er m in e L-Or n ith yl Am id e (30). The 4-nitrophenyl
ester of NR-tBoc-Nδ-Cbz-L-ornithine (from Sigma) was synthe-
sized via method A (65% yield). Coupling to spermine and
purification gave 69% of the protected conjugate. Deprotection
using hydrogenation (method B) and then acid treatment
(method A) gave 30 as its pentahydrochloride salt in 98% yield.
1H NMR (D2O, δ): 4.04 (t, 1H), 3.38 (m, 2H), 3.12 (m, 14H),
2.11 (m, 2H), 1.96 (m, 4H), 1.80 (m, 6H). 13C NMR (D2O,
ppm): 170.0, 53.6, 47.8, 47.7, 46.1, 45.4, 39.6, 37.4, 28.7, 26.3,
24.6, 23.7, 23.6, 23.4. HRMS: m/z calcd for C15H36N6O (M +
Na) 339.2848, found 339.2841.
N1-Sp er m in e L-Tr yp top h a n yl Am id e (32). N-tBoc-L-
tryptophan 4-nitrophenyl ester was obtained from Sigma and
coupled to spermine via method A (39% yield). Deprotection
by acid treatment gave 32 as its tetrahydrochloride salt in 92%
yield. 1H NMR (D2O, δ): 7.67 (d, 1H), 7.58 (d, 1H), 7.37 (s,
1H), 7.32 (t, 1H), 7.23 (t, 1H), 4.27 (t, 1H), 3.37 (m, 2H), 3.19
(m, 8H), 2.89 (t, 2H), 2.53 (m, 2H), 2.15 (m, 2H), 1.73 (m, 4H),
Gr ow th In h ibition Assa y. MDA-MB-231 cells were plated
in 96-well plates such that they would be in log growth for
the duration of the assay. The day after plating, drugs were
added to the cells, cells were grown for 6 days, and cell growth
was measured by MTS/PMS dye assay (Cell Titer 96 aqueous
nonradioactive cell proliferation assay; Promega, Madison,
WI). The assay was done in the presence of 1.0 mM AG and
1.0 µM SPD to ensure that any growth inhibition was not the
result of depletion of external polyamines in the medium. EC50
values represent the concentration of analogue that resulted
in 50% of the maximum growth inhibition achievable in the
presence of both DFMO and analogue.
P olya m in e An a lysis. MDA-MB-231 cells were seeded in
flasks such that they would be in log growth for 7 days. All
flasks received AG (1.0 mM), SPD (1.0 µM), and the appropri-
ate compound the day after seeding. After 6 days, cells were
harvested, washed, counted, and lysed in 0.40 M perchloric
acid. The HPLC method for the fluorometric detection of

Amino Acid/ Spermine Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3643
(18) DiPasquale, A.; White, D.; McGuire, J . Epidermal growth factor
stimulates putrescine transport and ornithine decarboxylase
activity in cultures human fibroblasts. Exp. Cell Res. 1978, 116,
317-323.
(19) Pohjanpelto, P. Putrescine transport is greatly increased in
human fibroblasts initiated to proliferate. J . Cell Biol. 1976, 68,
512-520.
(20) Volkow, N.; Goldman, S. S.; Flamm, E. S.; Cravioto, H.; Wolf,
A. P.; Brodie, J . D. Labeled putrescine as a probe in brain
tumors. Science 1983, 221, 673-675.
(21) Moulinoux, J .-Ph.; Quemener, V.; Khan, N. A. Biological sig-
nificance of circulating polyamines in oncology. Cell. Mol. Biol.
1991, 37, 773-783.
(22) Metcalf, B. W.; Bey, P.; Danzin, C.; J ung, M. J .; Casara, P.;
Vevert, J . P. Catalytic irreversible inhibition of mammalian
ornithine decarboxylase (EC 4.1.1.17) by substrate and product
analogues. J . Am. Chem. Soc. 1978, 100, 2551-2553.
(23) Bogle, R. G.; Mann, G. E.; Pearson, J . D.; Morgan, D. M. L.
Endothelial polyamine uptake: selective stimulation by L-
arginine deprivation or polyamine depletion. Am. J . Physiol.
1994, 266, C776-C783.
(24) Alhonen-Hongisto, L.; Seppa¨nen, P.; J a¨nne, J . Intracellular
putrescine deprivation induces uptake of the natural polyamines
and methylglyoxal bis(guanylhydrazone). Biochem. J . 1980, 192,
941-945.
(25) Persson, L.; Holm, I.; Ask, A.; Heby, O. Curative effect of d,l-2-
difluoromethylornithine on mice bearing mutant L1210 leuke-
mia cells deficient in polyamine uptake. Cancer Res. 1988, 48,
4807-4811.
(26) Sarhan, S.; Kno¨dgen, B.; Seiler, N. The gastrointestinal tract
as polyamine source for tumor growth. Anticancer Res. 1989, 9,
215-224.
dansylated polyamines from the lysates was based on the
procedure by P. M. Kabra et al.41 A detection level of 1.0 pmol
was achievable.
ODC Activity Assa y. This assay was slightly modified from
one previously described.62 MDA-MB-231 cells were seeded to
be in log growth for the duration of the experiment. The day
after seeding, compounds were added to the appropriate
flasks: 1 mM AG, 1.0 µM SPD, 100 µM Lys-Spm, 31, and 0.5
mM DFMO. After 3 days with the drugs the cells were
harvested by trypsinization, counted, and washed with PBS
prior to freezing. At the time of the assay, cells were thawed
in cold breaking buffer (25 mM Tris, 0.10 mM EDTA, 2.5 mM
DTT, at pH 7.5), homogenized, and frozen/thawed twice and
aliquots removed for protein determinations (Pierce Coomassie
protein assay). The homogenates were centrifuged at 10000
rpm for 10 min. A 50 µL sample of the supernatant was
combined with 200 µL of the reaction buffer containing 5 mM
Tris at pH 7.4, 0.4 mM L-ornithine, 40 µM pyridoxal-5-
phosphate, 5 mM DTT, and 1 µCi of l-[1-14C]ornithine (Ameri-
can Radiolabeled Chemicals, Inc., 55 mCi/mmol). The assay
mixture was incubated at 37 °C for 2 h in an Eppendorf tube
that was contained in a tightly capped 4 mL glass vial
containing 400 µL of 0.1 N NaOH. The reaction was stopped
by the addition of 125 µL of 10 M H2SO4. The 0.1 N NaOH in
the vial was counted by scintillation for radioactivity from
absorbed 14CO2 that was released from ornithine substrate.
Cell lysates were assayed in duplicate and were normalized
to protein concentration.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR
spectra of novel compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
(27) Hessels, J .; Kingma, A. W.; Ferwerda, H.; Keij, J .; van den Berg,
G. A.; Muskiet, F. A. Microbial flora in the gastrointestinal tract
abolishes cytostatic effects of alpha-difluoromethylornithine in
vivo. Int. J . Cancer 1989, 43, (6), 1155-1164.
(28) Bardocz, S.; Grant, G.; Brown, D. S.; Ralph, A.; Pusztai, A.
Polyamines in food; implications for growth and health. J .
Biochem. Nutr. 1993, 4, 66-71.
(29) Leveˆque, J .; Burtin, F.; Catros-Quemener, V.; Havouis, R.;
Moulinoux, J .-Ph. The gastrointestinal polyamine source deple-
tion enhances DFMO induced polyamine depletion in MCF-7
human breast cancer cells in vivo. Anticancer Res. 1998, 18,
2663-2668.
Refer en ces
(1) Cohen, S. S. A Guide to the Polyamines; Oxford University
Press: New York, 1998.
(2) Matthews, H. R. Polyamines, chromatin structure and transcrip-
tion. BioEssays 1993, 15, 561-566.
(3) Igarashi, K.; Kashiwagi, K. Polyamines: mysterious modulators
of cellular functions. Biochem. Biophys. Res. Commun. 2000, 271,
559-564.
(4) Ha, H. C.; Sirisoma, N. S.; Kuppusamy, P.; Zweier, J . L.; Woster,
P. M.; Casero, R. A. The natural polyamine spermine functions
directly as a free radical scavenger. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 11140-11145.
(30) Moulinoux, J . P.; Darcel, F.; Quemener, V.; Havouis, R.; Seiler,
N. Inhibition of growth of the U-251 human glioblastoma in nude
mice by polyamine deprivation. Anticancer Res. 1991, 11, 175-
180.
(31) Quemener, V.; Moulinoux, J. P.; Havouis, R.; Seiler, N. Polyamine
deprivation enhances antitumoral efficacy of chemotherapy.
Anticancer Res. 1992, 12, 1447-1454.
(32) Chamaillard, L.; Catros-Quemener, V.; Delcros, J . G.; Bansard,
J . Y.; Havouis, R.; Desury, D.; Commereuc, A.; Genetet, N.;
Moulinoux, J . P. Polyamine deprivation prevents the develop-
ment of tumour-induced immune suppression. Br. J . Cancer
1997, 76, 365-370.
(33) Krapcho, A. P.; Kuell, C. S. Monoprotected diamines. N-tert-
butoxylcarbonyl-R,ω-alkanediamines from R,ω-alkanediamines.
Synth. Commun. 1990, 20, 2559-2564.
(34) Blagbrough, I. S.; Geall, A. J . Practical synthesis of unsym-
metrical polyamine amides. Tetrahedron Lett. 1998, 39, 439-
442.
(35) Bodanszky, M.; Funk, K. W.; Fink, M. L. o-Nitrophenyl esters
of tert-butyloxycabonylamino acids and their application in the
stepwise synthesis of peptide chains by a new technique. J . Am.
Chem. Soc. 1973, 38, 3565-3570.
(36) Tsuji, M.; Nakajima, T.; Sano, I. Putrescine, spermidine, N-
acetylspermidine and spermine in the urine of patients with
leukaemias and tumors. Clin. Chim. Acta 1975, 59, 161-167.
(37) Aramaki, Y.; Yasuhara, T.; Higashijima; Yoshioka, M.; Miwa,
A.; Kawai, N.; Nakajima, T. 94. Chemical characterization of
spider toxin, J STX and NSTX. Proc. J pn. Acad. 1986, 62B, 359-
362.
(38) Asami, T.; Kagechika, H.; Hashimoto, Y.; Shudo, K.; Miwa, A.;
Kawai, N.; Nakajima, T. Acylpolyamines mimic the action of J oro
spider toxin (J STX) on crustacean muscle glutamate receptors.
Biomed. Res. 1989, 10, 185-189.
(5) Wolff, J . Promotion of Microtubule Assembly by Oligiocations:
Cooperativity between Charged Groups. Biochemistry 1998, 37,
10722-10729.
(6) Webb, H. K.; Wu, Z.; Sirisoma, N.; Ha, H.-C.; Casero, R. A.;
Woster, P. M. 1-(N-Alkylamino)-11-(N-ethylamino)-4,8-diazaun-
decanes: Simple Synthetic Polyamine Analogues That Differ-
entially Alter Tubulin Polymerization. J . Med. Chem. 1999, 42,
1415-1421.
(7) Carter, C. The Neuropharmacology of Polyamines; Academic
Press: San Diego, CA, 1994.
(8) Williams, K. Interaction of polyamines with ion channels.
Biochem. J . 1997, 325, 289-297.
(9) Ransom, R. W.; Stec, N. L. Cooperative modulation of [3H]MK-
801 Binding to the N-Methyl-D-Aspartate Receptor-Ion Channel
Complex by L-Glutamate, Glycine, and Polyamines. J . Neuro-
chem. 1988, 51, 830-836.
(10) Williams, K.; Romano, C.; Dichter, M. A.; Molinoff, P. B.
Minireview: Modulation of the NMDA receptor by polyamines.
Life Sci. 1991, 48, 469-498.
(11) Casero, R. A.; Woster, P. M. Terminally alkylated polyamine
analogues as chemotherapeutic agents. J . Med. Chem. 2001, 44,
1-26.
(12) J a¨nne, J .; Po¨so, H.; Raina, A. Polyamines in rapid growth and
cancer. Biochim. Biophys. Acta 1978, 473, 241-493.
(13) Scalabrino, G.; Ferioli, M. E. Polyamines in mammalian tumors.
Part I. Adv. Cancer Res. 1981, 35, 151-268.
(14) Scalabrino, G.; Ferioli, M. E. Polyamines in mammalian tumors.
Part II. Adv. Cancer Res. 1982, 36, 1-102.
(15) Seiler, N.; Dezeure, F. Polyamine transport in mammalian cells.
Int. J . Biochem. 1990, 22, 211-218.
(16) Seiler, N.; Dezeure, F. Polyamine transport in mammalian cells.
An update. Int. J . Biochem. Cell Biol. 1996, 28, 843-861.
(17) Khan, N. A.; Quemener, V.; Moulinoux, J .-P. Characterization
of polyamine transport pathways. In Neuropharmacology of
Polyamines; Carter, C., Ed.; Academic: San Diego, 1994; pp 37-
60.
(39) Iwasawa, H.; Kondo, S.; Ikeda, D.; Takeuchi, T.; Umezawa, H.
Synthesis of (-)-15-deoxyspergualin and (-)-spergualin-15-
phosphate. J . Antibiot. 1982, 35, 1655-1669.

3644 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Burns et al.
(40) Geall, A. J .; Taylor, R. J .; Earll, M. E.; Eaton, M. A. W.;
Blagbough, I. S. Synthesis of cholesteryl polyamine carbam-
ates: pKa studies and condensation of calf thymus DNA.
Bioconjugate Chem. 2000, 11, 314-326.
(41) Kabra, P. M.; Lee, H. K.; Lubich, W. P.; Marton, L. J . Solid-
phase extraction and determination of dansyl derivatives of
unconjugated and acetylated polyamines by reversed-phase
liquid chromatography: improved separation systems for
polyamines in cerebrospinal fluid, urine and tissue. J . Chro-
matogr. 1986, 380, 19-32.
(42) Kashiwagi, K.; Hosokawa, N.; Furuchi, T.; Kobayashi, H.;
Sasakawa, C.; Yoshikawa, M.; Igarashi, K. Isolation of polyamine
transport-deficient mutants of Escherichia coli and cloning of
the genes for polyamine transport proteins. J . Biol. Chem. 1990,
265, 20893-20897.
(43) 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.
(44) Sugiyama, S.; Vassylyev, D. G.; Matsushima, M.; Kashiwagi, K.;
Igarashi, K.; Morikawa, K. Crystal structure of PotD, the
primary receptor of the polyamine transport system in Escheri-
chia Coli. J . Biol. Chem. 1996, 271, 9519-9525.
(45) 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.
(46) Bergeron, R. J .; Feng, Y.; Weimer, W. R.; McManis, J . S.;
Dimova, H.; Porter, C.; Raisler, B.; Phanstiel, O. A comparison
of structure-activity relationships between spermidine and
spermine antineoplastics. J . Med. Chem. 1997, 40, 1475-1494.
(47) Porter, C. W.; 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.
(48) Huber, M.; Pelletier, J . G.; Torossian, K.; Dionne, P.; Gamache,
I.; Charest-Gaudreault, R.; Audette, M.; Poulin, R. 2,2′-Dithiobis-
(N-ethyl-spermine-5-carboxamide) is a high affinity, membrane-
impermeant antagonist of the mammalian polyamine transport
system. J . Biol. Chem. 1996, 271, 27556-27563.
(52) Li, Y.; MacKerell, A. D.; Egorin, M. J .; Bllesteros, M. F.; Rosen,
M.; Wu, Y.-Y.; Blamble, D. A.; Callery, P. S. Comparative
molecular field analysis-based predictive model of structure-
function relationships of polyamine transport inhibitors in L1210
cells. Cancer Res. 1997, 57, 234-239.
(53) 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, 65-77.
(54) Karahalois, P.; Mamos, P.; Vynios, D. H.; Papaioannou, D.;
Kalpaxis, D. L. The effect of acylated polyamine derivatives on
polyamine uptake mechanism, cell growth and polyamine pools
in Escherichia coli, and the pursuit of structure/activity relation-
ships. Eur. J . Biochem. 1998, 251, 998-1004.
(55) J aroszewski, J . W.; Matzen, L.; Frolund, B.; Krogsgaard-Laren,
P. Neuroactive polyamine wasp toxins: nuclear magnetic reso-
nance spectroscopic analysis of the protolytic properties of
philanthotoxin-343. J . Med. Chem. 1996, 39, 515-521.
(56) Moya, E.; Blagbrough, I. S. Syntheses and neuropharmacological
properties of arthropod polyamine amide toxins. In Neurophar-
macology of Polyamines; Carter, C., Ed.; Academic: San Diego,
1994; pp 167-184.
(57) Raditsch, M.; Geyer, M.; Kalbitzer, H. R.; J ahn, W.; Ruppersberg,
J . P.; Witzemann, V. Polyamine spider toxins and mammalian
N-methyl-D-aspartate receptors. Structural basis for channel
blocking and binding of argiotoxin636. Eur. J . Biochem. 1996,
240, 416-426.
(58) Tsubokawa, H.; Oguro, K.; Masuzawa, T.; Nakaima, T.; Kawai,
N. Effects of a spider toxin and its analoque on glutamate-
activated currents in the hippocampal CA1 neuron after is-
chemia. J . Neurophys. 1995, 74, 218-225.
(59) Weeks, R. S.; Vanderwerf, S. M.; Carlson, C. L.; Burns, M. R.;
O’Day, C. L.; Cai, F.; Devens, B. H.; Webb, H. K. Novel lysine-
spermine conjugate inhibits polyamine transport and inhibits
cell growth when given with DFMO. Exp. Cell Res. 2000, 261,
293-302.
(60) Devens, B. H.; Weeks, R. S.; Burns, M. R.; Carlson, C. L.; Brawer,
M. K. Polyamine depletion therapy in prostate cancer. Prostate
Cancer Prostatic Dis. 2000, 3, 275-279.
(49) 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.
(50) Minchin, R. F.; Martin, R. L.; Summers, L. A.; Ilett, K. F.
Inhibition of putrescine uptake by polypyridinium quaternary
salts in B16 melanoma cells treated with difluoromethylorni-
thine. Biochem. J . 1989, 262, 391-395.
(51) Aziz, S. M.; Gosland, M. P.; Crooks, P. A.; Olson, J . W.; Gillespie,
M. N. A novel polymeric spermine conjugate inhibits polyamine
transport in pulmonary artery smooth muscle cells. J . Pharma-
col. Exp. Ther. 1995, 274, 181-186.
(61) Nude mice were given an ip dose of 45 mg/kg three times daily
5 days a week for 14 days. No weight loss or toxicities were
noticed.
(62) Coleman, C. S.; Pegg, A. E. Assay of Mammalian Ornithine
Decarboxylase Activity Using [14C] Ornithine. In Methods in
Molecular Biology, Vol 79: Polyamine Protocols; Morgan, D., Ed.;
Humana Press, Inc.: Totowa, NJ , 1998.
J M0101040