Synthesis and biological evaluation of aminopolyamines

Article abstract of DOI:10.1021/jm050024m

Exploitation of the polyamine backbone as a vector for intracellular transport of various pharmacophores has focused largely on fixing the cargo molecule to one of the nitrogens in the linear chain. This communication describes the assembly of a model ami

Full text of DOI:10.1021/jm050024m

J. Med. Chem. 2005, 48, 3099-3102
3099
should be more favorable than when the cargo molecule
is negatively charged.1,15-17,25
Synthesis and Biological Evaluation of
Aminopolyamines
Recently, we have shown that it is possible to vector
iron chelators intracellularly via the polyamine trans-
port apparatus.26 A simple bidentate, neutral ligand,
1,2-dimethyl-3-hydroxypyridin-4-one (L1), was conju-
gated to spermine (SPM), generating 1-(12-amino-4,9-
diazadodecyl)-2-methyl-3-hydroxy-4(1H)-pyridinone. The
denticity of the iron complex was not affected by the
attachment of the SPM moiety. L1 does not achieve high
concentrations (∼1 µM) in cultured cells. In murine leu-
kemia L1210 cells, all of the indicators of intracellular
access, including (1) the effect on cell proliferation (IC50),
(2) the ability to compete with radiolabeled spermidine
for the polyamine transport apparatus (Ki), (3) the im-
pact on polyamine pools, and (4) effects on the polyamine
enzymes ODC, AdoMetDC, and SSAT, were consistent
with the polyamine fragment of the polyamine-chelator
conjugate serving as a vector. The adduct was trans-
ported quite efficiently into the cells; the cellular
concentration of 0.39 mM is more than 1900-fold higher
than the extracellular treatment concentration and is
also nearly 400 times higher than the cellular concen-
tration attained by the parent ligand L1. This vector
concept has now been successfully extended to the iron
chelator (S)-4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-meth-
yl-4-thiazolecarboxylic acid. The norspermidine conju-
gate (S)-4,5-dihydro-2-[2-hydroxy-4-(12-amino-5,9-diaz-
adodecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid
ethyl ester achieves very high intracellular concentra-
tions relative to its parent ligand (Bergeron et al.,
manuscript in press).
Exploitation of the polyamine backbone as a vector
has focused largely on attaching the cargo molecule to
one of the nitrogens in the linear chain, either a
terminal primary amine or a secondary internal nitro-
gen. The nature of the linkage between the nitrogens
and the cargo molecule is critical. The linkage can have
a profound impact on the molecule’s charge disposition
and thus its uptake properties. Clearly, an acyl or
sulfonyl linkage to the nitrogens prevents protonation
at physiological pH.
Consider the linear tetraamine SPM: acylation of a
single terminal nitrogen still leaves the requisite three
cationic SPD-like sites for binding and transport. How-
ever, acylation of a secondary nitrogen interrupts the
linear charge array. Although a triamine remains, the
systems looks more like a diamine insulated from a
monoamine. This problem could be overcome by intro-
ducing a primary amine into the polyamine backbone.
This primary amine could be linked via acyl or sulfonyl
fragments to a variety of cargo molecules without
affecting the charge substantially. The current work
describes (1) the assembly of such an aminopolyamine
pharmacophore, (2) an evaluation of the compound’s
effects on L1210 cell proliferation, (3) an evaluation of
its cellular uptake, and (4) its impact on polyamine pools
and enzymes by procedures that are standard in this
laboratory. The target chosen for this initial study was
a derivative of the well-characterized polyamine ana-
logueDESPM,6-amino-N1,N12-diethylspermine(6ADESPM,
Raymond J. Bergeron,* Guangfei Huang,§
James S. McManis, Hua Yao, and John Nhut Nguyen
Department of Medicinal Chemistry, University of Florida,
Gainesville, Florida 32610-0485
Received January 10, 2005
Abstract: Exploitation of the polyamine backbone as a vector
for intracellular transport of various pharmacophores has
focused largely on fixing the cargo molecule to one of the
nitrogens in the linear chain. This communication describes
the assembly of a model aminopolyamine analogue, 6-amino-
N1,N12-diethylspermine, and its biological properties. This
amino polyamine presents an additional site of attachment for
cargo molecules, reduces cell growth, and achieves cellular
concentrations that are higher than those of N1,N12-diethyl-
spermine.
It is now clear that N-alkylated polyamines exhibit
antineoplastic activity against a number of murine and
human tumor lines in vitro and in vivo.1,2 Although it
is well-established that these polyamine analogues
utilize the polyamine transport apparatus for incorpora-
tion into cells,3,4 deplete polyamine pools,5 drastically
reduce the level of ornithine decarboxylase (ODC)6,7 and
S-adenosylmethionine decarboxylase (AdoMetDC) ac-
tivities,8 and in some cases up-regulate spermidine/
spermine N1-acetyltransferase (SSAT),9-12 the precise
mechanism by which they induce death in tumor cells
still remains a mystery.13 A systematic structure-
activity study has shown that very small structural
alterations in these polyamine analogues can cause
pronounced changes in their biological activity at the
cellular and whole animal levels.5 This is exemplified
by the differences in how various analogues compete for
transport and how they influence key polyamine bio-
synthetic enzymes.
We have mapped out the structural boundary condi-
tions set by the polyamine transport apparatus on
polyamine analogues.1,2,5-8,11,14-19 Although steric issues
are important,1,15-17 charge is critical to recognition and
transport of these substrates.1,15,16,19-21 For example,
N1,N12-diethylspermine (DESPM), a tetracation at physi-
ological pH, competes well with spermidine (SPD) for
the polyamine transport apparatus and is concentrated
to millimolar levels intracellularly. N1,N12-Bis(2,2,2-
trifluoroethyl)spermine (FDESPM) is dicationic at pH
7.4 and competes poorly with SPD for the polyamine
transport apparatus. In the picture that emerges, the
polyamine transport apparatus provides biological coun-
teranions for the cationic nitrogens. Other investigators
have exploited these findings by attaching a variety of
cargo molecules to the spermine backbone.21-24 On the
basis of simple electrostatics, when a cargo molecule is
neutral or positively charged, binding to the transporter
* To whom correspondence should be addressed. Phone: (352) 846-
1956. Fax: (352) 392-8406. E-mail: bergeron@mc.cop.ufl.edu.
§ Present address: Alchem Laboratories Corporation, 13305 Rachael
Boulevard, Alachua, Florida 32615.
10.1021/jm050024m CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/02/2005

3100 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Letters
Table 1. L1210 Cell Growth Inhibition and Transport for
Alkylated Spermine Analogues
Scheme 1. Synthesis of the Aminopolyamine
6ADESPM
a The IC50 was estimated from growth curves for L1210 cells
grown in the presence of nine different extracellular concentrations
of drug spanning four logarithmic units: 0, 0.03, 0.1, 0.3, 1.0, 3,
10, 30, and 100 µM. IC50 data are presented as the mean of at
least two experiments with variation from the mean typically 10-
25% for the 96 h IC50 values. b Ki determinations were made by
following analogue inhibition of spermidine transport. All polyamine
analogues exhibited simple substrate-competitive inhibition of
[3H]SPD transport by L1210 cells. Values reported in the table
represent the mean of at least two or three experiments with a
variation typically less than 10%. c From ref 15. d From ref 16.
mercial availability of both stereoisomers of epoxide 3
lends itself nicely to assembling each enantiomer of
6ADESPM (1). This synthetic route is versatile in that
N-monoalkylsulfonamides besides 2 could be added to
epoxide 4. Furthermore, the primary amino group of 8
could be modified with alkyl, acyl, or other functionality
stable to mesitylenesulfonyl group removal, providing
access to analogues of 6ADESPM (1).
At present, it is unclear how the cell might see 6AD-
ESPM in terms of charge, i.e., as a tetracation or as a
fully protonated pentaamine. The pKa values for the
nitrogens remain to be determined. However, on the
basis of our previous pKa studies with the tetraamines
N1,N11-diethylnorspermine (DENSPM), DESPM (Table
1), and N1,N14-diethylhomospermine (DEHSPM),20
6ADESPM is likely to be a tetracation at physiological
pH. Although DEHSPM is largely tetracationic at
physiological pH, shortening the methylene backbone
betweenthechargedcenters(e.g.,DEHSPMvsDENSPM)
profoundly reduces the fraction of tetracation. The
shorter insulators between the cationic nitrogens and
the resulting electrostatic repulsion impede complete
protonation.32
The 48 and 96 h IC50 values (Table 1) suggest that
6ADESPM (8 µM at 48 h; 0.4 µM at 96 h) behaves more
like its tetracationic parent DESPM (30 and 0.2 µM at
48 and 96 h, respectively) than its dicationic analogue
FDESPM (>100 µM at 96 h).
The ability of 6ADESPM to compete with radiolabeled
SPD for uptake was compared to those of DESPM15 and
FDESPM16 (Table 1). The dication FDESPM was a poor
competitor (Ki ) 285 µM), behaving much like pu-
trescine (PUT). DESPM was an excellent competitor (Ki
) 1.6 µM). Interestingly, the pentaamine 6ADESPM (Ki
) 33.7 µM) does not bind to the transport apparatus
nearly as well as does DESPM, leaving questions about
the nature of the charge disposition.
1). Ensuing studies will focus on utilization of this vector
for intracellular transport of a variety of ligands.
The synthesis began with the alkylation of N,N′-bis-
(mesitylenesulfonyl)-N-ethyl-1,3-diaminopropane (2)19,28
with 4-bromo-1,2-epoxybutane (3)29,30 (Scheme 1). When
the anion of sulfonamide 2 (NaH in DMF) was treated
with an equimolar amount of 3 at room temperature,
selective displacement of bromide occurred,29 giving
epoxide 4 in 66% yield. When the anion of 2 (2 equiv)
was heated at 70 °C with ω-bromoepoxide 3, tetrasul-
fonamide alcohol 5 was generated in 80% yield. Reaction
of 5 with p-toluenesulfonyl chloride in pyridine and CH2-
Cl2 afforded secondary tosylate 6 in 61% yield. Displace-
ment of the hindered leaving group of 6 was effected
with potassium phthalimide in DMF at 85 °C, providing
fully protected pentaamine 7 in 62% yield. Phthalimide
7 was unreactive to prolonged heating with hydrazine
in CH3CH2OH. However, reduction of 7 with sodium
borohydride in aqueous 2-propanol and then heating the
mixture with acetic acid31 gave 6-amino-N1,N12-diethyl-
N1,N4,N9,N12-tetrakis(mesitylenesulfonyl)spermine (8)
in 74% yield. Removal of the mesitylenesulfonyl protect-
ing groups of 8 using HBr-acetic acid in phenol and
CH2Cl2 and conversion to the pentahydrochloride salt
furnished 6ADESPM (1) in 52% yield. It is important
to point out that this product is racemic. The synthetic
methodology of Scheme 1 in conjuction with the com-
L1210 cells were treated with DESPM or 6ADESPM
at about 5 times their respective 48 h IC50 values15
(Table 2) to ensure that we are working in a range
where growth inhibition is occurring. At 150 µM in the
culture media, DESPM depleted PUT and SPD to 0%

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3101
Table 2. Effect of Alkylated Spermine Analogues on
A highly flexible synthetic method is now available
for the assembly of aminopolyamines. Because of the
observed biological profile of 6ADESPM (1), a racemic
mixture, each enantiomer will be assembled and evalu-
ated. The key synthon for further studies, the tet-
ramesitylenesulfonamide with its single free amine (8),
will allow coupling of various cargo molecules without
significantly compromising charge distribution. The
primary amine can be acylated, and protecting mesityl-
enesulfonyl groups can then be removed, leaving a
vector with four cationic centers.
The resulting 6ADESPM behaves very much like its
parent drug, slowing cell growth, competing effectively
with SPD for the polyamine transport apparatus, reduc-
ing polyamine pools and the activities of ODC and
AdoMetDC, and stimulating SSAT. Thus, it would seem
that introduction of an amino group into the polyamine
backbone of DESPM is compatible with cellular uptake.
In fact, this aminopolyamine achieves cellular concen-
trations that are almost 2 times higher than those of
DESPM itself. The question as to whether there is
anything patently unusual about the biological proper-
ties of this model vector itself has now been answered.
Polyamine Pools in L1210 Cells
treatment
compd
concn (µM) PUTa SPDa SPMa cellular concnb
DESPMc
150
500
50
0
87
26
0
94
7
22
116
32
1.78
<0.02
3.10
FDESPMc
6ADESPM
a Putrescine (PUT), spermidine (SPD), and spermine (SPM)
levels after 48 h of treatment are given as percent of the polyamine
found in untreated controls. The control values in pmol/106 L1210
cells are PUT ) 183 ( 15, SPD ) 2694 ( 232, SPM ) 774 ( 81.
b The cellular concentration of the analogue is expressed as nmol/
106 cells. Untreated L1210 cells (106) correspond to about 1 µL
volume; therefore, the concentration can be estimated as mM.
c From ref 16.
Table 3. Impact of Alkylated Spermine Analogues on
Ornithine Decarboxylase (ODC), S-Adenosylmethionine
Decarboxylase (AdoMetDC), and Spermidine/Spermine
N1-Acetyltransferase (SSAT) in L1210 Cellsa
compd
ODC
AdoMetDC
SSAT
DESPMb
3
28
460
FDESPMc
6ADESPM
100
100
97
16 ( 2
58 ( 8
667 ( 16
a Enzyme activity is expressed as percent of untreated control
for ODC (1 µM at 4 h), AdoMetDC (1 µM at 6 h), and SSAT (10
µM at 48 h). Each experiment included a positive control that had
a known, reproducible impact on enzyme activities (mean ( SD):
1 µM DEHSPM lowered ODC to 6.7 ( 2.6% of the untreated
control; 1 µM DEHSPM decreased AdoMetDC to 40.7 ( 6.2% of
the untreated control; 2 µM DENSPM increased SSAT to 1029 (
87% of the untreated control. Data shown in the table represent
the mean of at least three experiments and have variances
consistent with those suggested by the positive control data
presented above. b From ref 15. c From ref 16.
Acknowledgment. Funding was provided by the
National Institutes of Health Grant No. R01-DK49108.
We thank April M. Franklin for her technical aid and
Dr. Eileen Eiler-McManis for her editorial and organi-
zational assistance.
Supporting Information Available: Experimental pro-
cedures and analytical and spectral data for 6ADESPM. This
material is available free of charge via the Internet at http://
pubs.acs.org.
of control values, reduced SPM to 22% of control, and
accumulated to a cellular concentration of 1.78 mM.16
The polyamine pools of cells treated with 50 µM
6ADESPM were affected far less; PUT, SPD, and SPM
were diminished to 26, 7, and 32% of control values,
respectively. However, 6ADESPM achieved a level
nearly 2 times that of DESPM, 3.10 mM. Although the
extracellular treatment concentration of FDESPM (500
µM)16 was nearly twice its Ki value, the cellular con-
centration was <2% that of DESPM and <0.6% that of
6ADESPM; polyamine pools remained virtually un-
changed.
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On a comparative basis, the results (Table 3) are not
unexpected and are consistent with the effects of the
drug on polyamine pools. The compound with the
highest IC50, the highest Ki, and the least effect on
polyamine pools, FDESPM, also has no impact on the
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