Polyamine transport inhibitors: Design, synthesis, and combination therapies with difluoromethylornithine

Article abstract of DOI:10.1021/jm401174a

The development of polyamine transport inhibitors (PTIs), in combination with the polyamine biosynthesis inhibitor difluoromethylornithine (DFMO), provides a method to target cancers with high polyamine requirements. The DFMO+PTI combination therapy results in sustained intracellular polyamine depletion and cell death. A series of substituted benzene derivatives were evaluated for their ability to inhibit the import of spermidine in DFMO-treated Chinese hamster ovary (CHO) and L3.6pl human pancreatic cancer cells. Several design features were discovered which strongly influenced PTI potency, sensitivity to amine oxidases, and cytotoxicity. These included changes in (a) the number of polyamine chains appended to the ring system, (b) the polyamine sequence, (c) the attachment linkage of the polyamine to the aryl core, and (d) the presence of a terminal N-methyl group. Of the series tested, the optimal design was N¹,N^1′,N^1″-(benzene-1,3, 5-triyltris(methylene))tris(N⁴-(4-(methylamino)butyl)butane-1,4- diamine, 6b, which contained three N-methylhomospermidine motifs. This PTI exhibited decreased sensitivity to amine oxidases and low toxicity as well as high potency (EC₅₀ = 1.4 μM) in inhibiting the uptake of spermidine (1 μM) in DFMO-treated L3.6pl human pancreatic cancer cells.

Full text of DOI:10.1021/jm401174a

Article
pubs.acs.org/jmc
Polyamine Transport Inhibitors: Design, Synthesis, and Combination
Therapies with Difluoromethylornithine
Aaron Muth,† Meenu Madan,‡ Jennifer Julian Archer,‡ Nicolette Ocampo,† Luis Rodriguez,†
and Otto Phanstiel, IV*,‡
†Department of Chemistry, University of Central Florida, 4000 Central Florida Boulevard, Orlando, Florida 32816-2366, United
States
‡Department of Medical Education, 12722 Research Parkway, University of Central Florida, Orlando, Florida 32826-3227, United
States
S
* Supporting Information
ABSTRACT: The development of polyamine transport inhibitors (PTIs), in
combination with the polyamine biosynthesis inhibitor difluoromethylornithine
(DFMO), provides a method to target cancers with high polyamine requirements.
The DFMO+PTI combination therapy results in sustained intracellular polyamine
depletion and cell death. A series of substituted benzene derivatives were evaluated for
their ability to inhibit the import of spermidine in DFMO-treated Chinese hamster
ovary (CHO) and L3.6pl human pancreatic cancer cells. Several design features were
discovered which strongly influenced PTI potency, sensitivity to amine oxidases, and
cytotoxicity. These included changes in (a) the number of polyamine chains appended to the ring system, (b) the polyamine
sequence, (c) the attachment linkage of the polyamine to the aryl core, and (d) the presence of a terminal N-methyl group. Of
the series tested, the optimal design was N1,N1′,N1″-(benzene-1,3,5-triyltris(methylene))tris(N4-(4-(methylamino)butyl)butane-
1,4-diamine, 6b, which contained three N-methylhomospermidine motifs. This PTI exhibited decreased sensitivity to amine
oxidases and low toxicity as well as high potency (EC50 = 1.4 μM) in inhibiting the uptake of spermidine (1 μM) in DFMO-
treated L3.6pl human pancreatic cancer cells.
treated cells often up-regulate polyamine transport activity to
replenish their depleted intracellular polyamine pools. This
shortcoming of DFMO therapy has prompted the development
of polyamine transport inhibitors (PTIs) in an attempt to block
the compensatory response of up-regulated polyamine import.
Prior studies by Ask et al. in 1992 demonstrated that DFMO
modestly increased the survival time of mice treated with
murine leukemia L1210 cells and a low polyamine diet.5 In
contrast, DFMO provided 30−75% cures in leukemic mice
bearing mutant L1210-MGBGr cells, which were deficient in
polyamine transport.5 This work provided evidence that
inhibition of polyamine transport by genetic means could
potentiate the effects of DFMO and provide cures. In theory,
such an outcome can also be obtained using DFMO in
combination with a small molecule which acts as polyamine
transport inhibitor (PTI).
Indeed, the combination therapy of DFMO+PTI can be very
effective in depleting intracellular polyamine levels and limiting
tumor growth. For example, Burns et al have demonstrated the
utility of a lysine-spermine conjugate (D-Lys-Spm, MQT 1426,
1a, Figure 1) in combination with DFMO, in vitro.6 The D- and
L- enantiomers (1a and 1b) gave similar performance in vitro.7
This combination therapy was also demonstrated in cats with
oral squamous cell carcinoma.8 In 2009, these investigators
INTRODUCTION
■
Polyamines are essential growth factors for cells and play key
roles in transcription, translation, and chromatin remodeling.1
Intracellular polyamine homeostasis is maintained by a balance
between polyamine biosynthesis, degradation and transport.1d
The polyamine transport system (PTS) is an important target,
as many cancer cell types readily import polyamines to sustain
their growth rate, especially in the presence of polyamine
biosynthesis inhibitors such as difluoromethylornithine
(DFMO).1a−d,2 Numerous tumor types have been shown to
contain elevated polyamine levels and have active transport
systems to import exogenous polyamines.1a These cancer cell
properties facilitate the cell-selective delivery of polyamine−
drug conjugates to cancer cell types.1a−d,2
Many cancer cell types have been shown to have high
intracellular polyamine levels, which are acquired via either
polyamine biosynthesis or import pathways. A balance is
maintained between these two processes, and there are cellular
feedback mechanisms to guard against intracellular polyamine
overload (e.g., antizyme induction).1e One of the key enzymes
involved in polyamine biosynthesis is ornithine decarboxylase
(ODC). For cancer cells, ODC is often up-regulated in an
effort to increase the intracellular pools of polyamines via the
biosynthetic pathway, and ODC itself is considered a proto-
oncogene.3 Early efforts to control cell growth led to the
development of DFMO as a suicide inhibitor of ODC.4a−c
Despite the success of DFMO as an ODC inhibitor, DFMO-
Received: August 1, 2013
Published: January 9, 2014
© 2014 American Chemical Society
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Figure 1. Structures of polyamine-transport-targeting compounds 1−8.
reported significant improvements in potency by adding a C16
lipophilic substituent to their design (1c).7 This improved PTI
(1c) was used in combination with DFMO in the treatment of
squamous cell carcinomas (SCC) in a transgenic ODC mouse
model of skin cancer, and 88% of SCCs showed complete or
nearly complete remission. These promising results suggested
that further optimization of the DFMO+PTI strategy was
warranted.
A major impediment to PTI design, however, is our limited
knowledge of the mammalian polyamine transport system itself.
While genes associated with polyamine transport have been
described in lower organisms (e.g., E. coli and C. elegans), the
genes involved in mammalian transport are only now being
identified.9 As result, most of our understanding of the PTS
stems from phenotypical observations, relative kinetic uptake
studies, and structure−activity relationships.
Two models of mammalian polyamine transport have been
proposed by Poulin10 and Belting11 and were recently updated
by Gerner to include findings with Caveolin-1, nitric oxide
synthase 2 (NOS2), and SLC3A2.9f Poulin suggested that
polyamines enter the cell through an active plasma membrane
transporter, followed by sequestration into polyamine-seques-
tering vesicles (PSVs).10b In order for polyamines to internalize
within these PSVs, a vesicular H+/polyamine carrier is needed
to facilitate the import and escape from the PSV.10b Belting, on
the other hand, suggested a multistep endocytotic process
where polyamines bind to heparan sulfate proteoglycans in
caveolae.11b Once bound, the polyamines are then endocytosed
via a caveolin-dependent process and their heparan sulfate
chains are subsequently cleaved and further processed by NO
to liberate the polyamines.11b Heparan sulfate is a proteoglycan
with an architecture that has carboxylate and sulfate groups
separated by discrete distances. This is consistent with known
observations where the number of charges and distances
separating the cationic charges on the polyamine substrate all
influence polyamine-binding events and uptake.2c−e,12 These
two models are not mutually exclusive and may operate in
tandem or in concert to maintain polyamine homeostasis.
Beyond these two models, it is currently unclear how the
recently discovered genes9g involved in mammalian polyamine
transport operate and how they control these or other
polyamine uptake processes. Indeed, this area is ripe for further
investigation, and the development of potent PTIs which target
specific or several polyamine transport pathways will be of great
value to investigators interested in polyamine-transport
processes and further exploration of the existing models. The
present study explores the potency and performance of PTIs
with multiple polyamine arms with the goal to provide new
molecular tools for investigating polyamine transport.
The polyamine transport system (PTS) has shown wide
structural tolerance for importing N-substituted-polyamine
derivatives. Monosubstituted systems such as 2−4 were
effective PTS ligands; however, disubstituted analogues such
as 5a were more superior PTS ligands (Figure 1).2c−e,g,13 In this
regard, compounds which contain preferred polyamine
sequences often readily interact with the PTS.2c,d Recent
work has also shown that the number of appended polyamine
chains is important. For example, the disubstituted benzene
derivative 5a is a potent transport ligand and is highly toxic,
whereas the trisubstituted derivative 6a is a potent PTI and is
relatively nontoxic.
Compounds 5a and 6a illustrate two scaffolds, which can
display PTI activity. First, a selective PTS ligand such as 5a with
high affinity for the PTS can readily out-compete the native
polyamines for the putative cell surface receptors and
theoretically function as an effective PTI. The transported
ligand 5a can also enter and kill the cell. The toxicity of 5a,
however, becomes dose-limiting for in vitro experiments
designed to assess the PTI activity of 5a. Disubstituted
analogue 5a had a low Ki value in L1210 murine leukemia
cells (0.52 μM) and a L1210 IC50 value of 0.16 μM. Even
though compounds such as 5a which target, enter, and kill cells
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a
Scheme 1.
a
Reagents: (a) K2CO3, Aliquat, CH2Cl2/H2O; (b) 4 M HCl, EtOH.
via the PTS have clear value, they were undesirable as PTIs
because of high toxicity.13b
The impetus for making triamide 7c was based upon the fact
that the planned amide examples, 7a and 7b, incorporated non-
native polyamine sequences (e.g., homospermidine). We
speculated that the use of the native polyamine, spermine,
could provide a triamide-containing PTI (7c) with lower
toxicity. Indeed, significant changes in toxicity have been
observed in derivatives containing subtle changes in their
polyamine sequence.13a The new conjugates 7a−c were also
synthesized to better understand how the attachment of the
polyamine to the aryl core affected toxicity and potency.
As shown in Scheme 1, synthesis of these novel PTIs (7a−c)
required the synthesis of the known Boc-protected polyamines
(10a−c).2d,16,17 While 10a and 10b were generated in
successive steps from 4-bromobutyronitrile and mono-Boc-
protected putrescine using established methods,16 the spermine
adduct was made by an alternative route directly from spermine
using salicylaldehyde (1 equiv) as shown in Scheme 2.18
Our goal was to develop PTIs with low toxicity, which could
ultimately be given chronically to patients in combination with
DFMO. The trisubstituted derivative 6a illustrates a second
paradigm, which involves the use of a ligand with high affinity
for the polyamine transport system with low cellular toxicity.
This type of design is preferred, as one avoids the toxicity
constraints of 5a. For example, 6a had virtually the same Ki
value as 5a in L1210 murine leukemia cells (5a: 0.52 μM, 6a:
0.49 μM) but a dramatically higher L1210 IC50 value (5a: 0.16
μM, 6a: 122 μM). In short, the trisubstituted system 6a was
over 760-fold less toxic than the disubstituted system 5a.
Recent efforts with 6a also demonstrated its high IC50 values
(IC50 > 100 μM) in CHO and CHO-MG cells.13b Compound
6a also blocked the uptake of a PTS-selective probe, Ant44 (2),
into CHO cells.13b These promising findings for 6a provided
the impetus for the current study to further improve its design.
While the area of PTI development has recently been reviewed
in detail,14 this report describes our efforts to develop nontoxic
PTIs predicated upon the novel trisubstituted design of 6a.
a
Scheme 2
RESULTS AND DISCUSSION
■
The observation that trisubstituted 6a was relatively nontoxic
PTI whereas the 1,4-disubstituted system 5a was a toxic PTS
ligand led to key questions which needed to be addressed.
Which structural component of 6a was responsible for its low
toxicity: (a) the 1,3-substitution pattern or (b) the presence of
the third polyamine substituent? These questions were
addressed via the synthesis and bioevaluation of the triamide
systems 7a−c (Figure 1) and the meta-substituted benzene ring
compounds (8a and 8b). Using these model systems, we were
able to clearly demonstrate that the third polyamine substituent
was responsible for the low toxicity of 6a. This insight was then
extended to include the N-methyl derivative 6b, which had even
lower toxicity, decreased sensitivity to amine oxidases, and
potency similar to that of 6a. In summary, these studies
provided a nontoxic, potent PTI 6b for future use in
combination with DFMO in vivo.
a
Reagents: (a) 2-Hydroxybenzaldehyde; (b) di-tert-butyl dicarbonate;
(c) CH3ONH2/Na2CO3 (45% yield after three steps).
Synthesis. The synthesis and characterization of 1,3,5-
trisubstituted derivative 6a has been previously described by
Kaur et al.,13b and the synthesis of L-lysine−spermine conjugate
1b was previously described by Burns et al.15 These
compounds, 1b and 6a, were resynthesized for comparison
purposes in vitro with the new PTIs.
Formation of the salicylimine occurred at the primary amine
sites. This allowed for easy introduction of Boc groups to the
remaining three amine centers of spermine in situ to give 13.
Subsequent hydrolysis of the imine 13 with methoxyamine gave
the desired tri-Boc-protected spermine 10c in 45% yield after
three steps. This process has significant advantages in yield and
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a
Scheme 3
a
Reagents: (a) 25% MeOH/CH2Cl2, Boc-protected amine 10a or 10b; (b) 50% MeOH/CH2Cl2, NaBH4; (c) 4 M HCl, EtOH.
a
time over other methods due to the availability of spermine
versus the multistep construction of the 3,4,3-triamine
sequence using omega bromoalkanonitriles and acrylonitriles.19
Each of the three amines 10a−c were then coupled to the
commercially available 1,3,5-benzenetricarboxylic acid chloride
9 under biphasic conditions to give the respective triamides
11a−c in moderate yield (56%, 75%, and 40%, respectively,
Scheme 1). Triamides 11a−c were then deprotected to give the
respective compounds 7a−c in high yield, respectively.
Disubstituted derivative 8a was generated to determine if the
third polyamine substituent present in 6a was necessary for
PTS inhibition and provided a control to determine whether
meta-substitution contributed to the reduced toxicity observed
with 6a (via comparisons with 8a and 5a). In addition, 8b was
synthesized to evaluate how extending the polyamine sequence
and number of charges presented to the putative cell-surface
receptor affected PTI performance. The two-arm platforms
were synthesized from the starting 1,3-dialdehyde 14, which
was commercially available.13b As shown in Scheme 3, the
synthesis began with the reductive amination of aldehyde 14
with the corresponding protected polyamine (10a or 10b) to
yield compounds 15a and 15b, respectively.16 Deprotection
then gave the meta-substituted systems 8a and 8b in 44% and
51% overall yield after two steps, respectively.
Scheme 4
The above compounds were generated for comparisons to
the known PTIs 6a and 1b. In addition, prior work in our lab
with 2 and its N-methyl analogue 3 demonstrated that N-
methylation led to polyamine drugs with lower toxicity and
improved stability toward amine oxidases.17a This same trend
was also recently observed in the related disubstituted systems,
e.g., 5a vs 5b.13b Therefore, we elected to also apply the N-
methylation technology to our trisubstituted PTI design in an
effort to generate a PTI 6b with lower toxicity and improved
stability.
As shown in Scheme 4, the synthesis of 6b began with the
synthesis of triol 17 from the commercially available triester 16
using LiAlH4. Attempts to oxidize triol 17 to aldehyde 18 with
pyridinium dichromate (PDC) at room temperature gave
incomplete reactions. IBX was found to be superior in this
regard and gave 79% yield after both steps (reduction/
oxidation).20 Subsequent coupling of aldehyde 18 with the
known Boc-protected N1-methylhomospermidine derivative
1913b gave trisubstituted adduct 20b (39%) which was then
converted to 6b (85%) with 4 M HCl. Note: the related 6a was
made from 20a by a similar route using 18 and 10a.13b The
yields obtained for 6a (71%) and its intermediate 20a (47%)
during their resynthesis13b are listed in Scheme 4 for
completeness. The synthesis of the starting amines 10a and
19 have been previously described.16,13b
a
Reagents: (a) LiAlH4/THF; (b) IBX/tert-butyl alcohol; (c) 18 in
25% MeOH/CH2Cl2; (d) 50% MeOH/CH2Cl2/NaBH4; (e) 4 M
HCl/EtOH.
Bioevaluation of these compounds was then conducted using
a series of assays designed to compare the ability of each
compound to target the PTS of mammalian cells.
CHO and CHO-MG* Studies. Chinese hamster ovary
(CHO) cells were chosen along with a mutant cell line (CHO-
MG*) to comment on how the synthetic conjugates gain access
to cells.2c−e,21 The CHO-MG* cell line is polyamine-transport
deficient and represents a model for alternative modes of entry
(other than the PTS) including passive diffusion or use of
another transport system. The wild-type CHO cell line, on the
other hand, represents cells with high polyamine transport
activity.22 A comparison of toxicity in these two CHO cell lines
provided a screen that would detect selective use of the PTS.
High utilization of the PTS by the polyamine compounds
would be very toxic to CHO cells. However, reduced toxicity
would be expected in CHO-MG* cells due to its limited
transport activity.2c A CHO-MG*/CHO IC50 ratio was
determined for each compound, and a high CHO-MG*/
CHO ratio was expected for highly PTS-selective compounds.
The results for this screen are listed in Table 1. The published
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a e
‑
Table 1. Biological Evaluation of Polyamine Derivatives (1b, 5a, 6−8) in CHO and CHO-MG* Cells
b
c
d
compound
CHO IC10 (μM)
CHO EC50 (μM)
CHO-MG* IC50 (μM)
CHO IC50 (μM)
CHOMG*/CHO IC50 ratio
1b (L-Lys-Spm)
5a (44Bn44)
>100
0.001
10
1.37
NA
0.34
0.42
60
>100
>100
ND
727
ND
ND
ND
4.3
19.6 ( 0.8)
66.9 ( 6.2)
0.027 ( 0.001)
>100
6a (Trimer44)
6b (Trimer44NMe)
7a (Triamide44)
7b (Triamide444)
8a (mBn44)
e
10
>100
>100
>100
1
>100
>100
NA
NA
NA
69.1 ( 4.0)
30.0 ( 0.8)
13.4 ( 0.8)
16.0 ( 0.7)
0.028 ( 0.002)
0.01
0.01
1072
335
8b (mBn444)
0.04 ( 0.007)
a
b
CHO and CHO-MG* cells were incubated with 1 mM AG for 24 h prior to compound addition. The IC10 value represents the highest
concentration of compound which resulted in ≤10% cell growth inhibition (i.e., ≥ 90% cell viability) vs an untreated control over the same time
c
period (48 h). The EC50 value is the concentration of the PTI compound needed to reduce the cell viability halfway between the % viability
observed with DFMO+Spd control and the % viability observed for the DFMO-only control (DFMO IC50 CHO: 4.2 mM). (Note: these are
illustrated in Figure 3 as the green and red lines, respectively). These values are listed here in Table 1 for completeness and comparison to the other
properties observed in CHO cells. The listed value is the EC50 value for the respective PTI in its corresponding DFMO+Spd+PTI experiment in
d
e
CHO cells. A high CHO-MG*/CHO IC50 ratio indicates a compound with high utilization of the PTS to enter and kill cells. The CHO-MG* IC50
of 6b was 121.7 ( 3.9) μM. Note: compound 7c was not evaluated in CHO but was evaluated in L3.6pl cells (Table 2). Note: Cells were incubated
for 48 h at 37 °C in 5% CO2 with the respective conjugate. All experiments were in triplicate. ND: not determined because of low toxicity in both
CHO cell lines. NA: not applicable.
PTI (Lys-Spm, 1b) was synthesized and included as a positive
control.6,7,15,23
derivative 5a was very toxic (CHO IC50: 0.027 μM). Thus, 6a
and 6b were found to have >3700 fold lower toxicity in CHO
cells than 5a. A similar result was found previously in murine
leukemia L1210 cells, where 6a was found to be >760 fold less
toxic than 5a in L1210 cells (L1210 IC50: 6a = 122.3 μM and
5a = 0.16 μM). These two compounds, however, possessed
nearly identical Ki values in L1210 cells (5a: 0.52 μM 0.11
and 6a: 0.49 μM 0.02, respectively),13b indicating that these
two compounds bind to the L1210 PTS recognition elements
with nearly equal affinity.
Given the dramatic differences in toxicity and similar binding
affinities of 5a and 6a, we speculated that only two of the
polyamine arms may interact with the putative cell surface
receptor. This idea was investigated via additional studies to
test whether the third polyamine arm or the meta substitution
pattern of 6a was responsible for the lower toxicity of 6a
compared to 5a.
The control compound 8a, which contained two polyamine
arms in a 1,3-substitution pattern, provided a model system to
address this issue. If the third polyamine arm was required for
low toxicity, then the two-arm moiety 8a would be a potent
PTS targeting compound with high toxicity. If a meta
substitution pattern was critical for low toxicity, then 8a
would have low toxicity. Of these two possible outcomes, we
found the former to be the case.
Indeed, evaluation in CHO and CHO-MG* cells proved to
be very interesting. Compound 8a had nearly identical toxicity
as 5a (CHO IC50 8a: 27 nM; 5a: 28 nM) and 8a was the most
PTS selective of the series (e.g., CHOMG*/CHO IC50 ratio:
1072 in Table 1). This finding strongly suggested that the third
polyamine arm of 6a was in large part responsible for the low
toxicity of 6a in CHO cells. Note: 6a had >3500-fold lower
toxicity than 8a in CHO cells.
The CHO-MG* line was derived from the original CHO-
MG line obtained from Flintoff et al.22a,24 and provided similar
results with numerous previously synthesized controls.17b For
example, the respective CHO-MG/CHO IC50 ratios for
previously synthesized polyamine-anthryl conjugates, 2 and
5a were 14812 and 677,13b whereas the CHO-MG*/CHO IC50
ratios of 2 and 5a were 43 and 727, respectively.17b As the
original stocks of CHO-MG were no longer available from their
original source, CHO-MG* were used in the PTS screen.
As shown in Table 1, the IC10 value of each compound was
determined in CHO cells. The IC10 value represents the highest
concentration of compound which resulted in ≤10% cell
growth inhibition (i.e., ≥90% cell viability) vs an untreated
control over the same time period. At its IC10 value, the
compound was deemed essentially nontoxic. The IC10 value
provided the highest dose at which the PTI could be added to
the cell culture and not result in a significant cytotoxic response
(due to its own intrinsic toxicity). This was important as the
assay used to assess PTI function measured decreases in cell
viability.
The IC50 value was the concentration of the compound
which resulted in 50% cell growth inhibition vs an untreated
control over the same time period. For example, a compound
with a low IC50 value (e.g., 5a: CHO IC50=0.027 μM) was
considered to be more toxic to CHO cells than 6a (IC50>100
μM). The IC50 values for each compound were determined in
CHO and CHO-MG* cells, and the CHO-MG*/CHO IC50
ratio was used to assess the ability of the compound to enter
and inhibit growth of the cell via the PTS. PTS-selective
compounds such as 5a, 8a, and 8b had high CHO-MG*/CHO
IC50 ratios and were considered very selective in entering and
inhibiting the growth of cells with active polyamine transport
systems. These compounds were also competitive inhibitors of
polyamine transport, and exogenous spermidine has been
shown to rescue cells treated with 5a.17b While these
compounds may be of interest for targeting cancers via their
PTS, the goal here was to identify nontoxic PTIs and not lethal
PTS-targeting compounds.17b
The tetraamine analogue 8b was found to be less PTS
selective (CHO-MG*/CHO IC50 ratio: 335) than its counter-
part, 8a (IC50 ratio: 1072). This demonstrated that the longer
polyamine message lowered the PTS selectivity of the design.
This finding was also observed in the comparison of
monosubstituted triamine 2 and tetraamine 4 (Figure 1),
where triamine 2 was more PTS selective than tetraamine 4 in a
related CHO/CHO-MG screen.2d Indeed, triamine derivatives
were found to be superior to tetraamines in terms of binding
and entering cells via the PTS.13a
The 1,3,5-trisubstituted analogues 6a and 6b exhibited very
high IC50 values in CHO cells (>100 μM) and were deemed
relatively nontoxic. In stark contrast, the 1,4-disubstituted
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A comparison of 8a (meta) and 5a (para) suggested that the
meta substitution pattern enhanced the PTS targeting ability of
the drug. This is observed in the higher CHO-MG*/CHO ratio
for 8a in Table 1. We hypothesized that this was due to the
intrinsic preorientation of the appended polyamine arms in the
meta-substituted system 8a. Although the effect was cell line
dependent, other authors have demonstrated that conforma-
tional constraint of the linker connecting two polyamine chains
can impart significant differences in potency in PC-3, MCF7,
and U251MG cells.25 Therefore, we concluded that the
polyamine sequence, the number of polyamine chains, and
the orientation of these chains all influenced the PTS-targeting
ability of these compounds and their associated toxicity profiles.
Compound 7a presented a unique opportunity to evaluate a
1,3,5-substituted triamide platform as a PTI. The amide linkage
in 7 removes the formal positive point charge at the ring
attachment position. From the cationic charge perspective,
triamides 7a and 7b represent architectures, which present
three putrescine (7a) or three homospermidine (7b)
“messages” to the putative cell surface receptors, respectively.
The trends in compound toxicity were very insightful. As
shown in Table 1, the IC50 of 7a was >100 μM, whereas the
IC50 of 7b was 16 μM. In the comparative CHO and CHO-
MG* screen, triamide 7b demonstrated modest PTS selectivity
(IC50 ratio: 4.3) and the ratio for 7a was not determined due to
its low toxicity in both cell lines (Table 1).
transport activity of DFMO-treated CHO cells was used to
assess the PTI activity of the new compounds. Specifically, we
investigated the ability of each compound to block the entry of
a rescuing dose of spermidine (1 μM) in DFMO-treated CHO
cells.15,23 This method required the determination of three
parameters before running the definitive assay. First, the 48 h
IC50 value of DFMO was determined in CHO cells (DFMO 48
h IC50 in CHO: 4.2 mM). Second, CHO cells were treated with
the IC50 dose of DFMO, and the minimum amount of
spermidine (Spd) needed to rescue these DFMO-treated cells
back to their maximum viability (>90% viability in this cell line)
was determined. In CHO cells, spermidine at 1 μM was
sufficient to rescue the DFMO-treated CHO cells. Third, the
48 h IC10 value was determined for each compound. The first
two parameters defined the DFMO and Spd concentrations to
be used, and these remained fixed throughout the assay. The
third parameter (IC10 value) defined the upper limit of the
concentration of the PTI agent that could be used in the
DFMO+Spd+[PTI] experiment without introducing significant
toxicity from the PTI agent itself to the DFMO+Spd-treated
cells. Control experiments with Spd and PTI (without DFMO)
revealed that no synergistic toxicity was induced by these two
polyamine compounds when dosed together in CHO cells. In
this manner, we could be confident that the results obtained
were due to the ability of the compound to block spermidine
entry and was not due to PTI toxicity or unexpected Spd+PTI
toxicity. The fact that the active compounds had EC50 values
significantly below the compound’s IC10 value provided further
support for this approach.26
We found that PTI potency was best evaluated through the
use of spermidine (Spd). Spermidine was found to effectively
rescue L1210 and CHO cells from polyamine-based drugs and
DFMO.2g We noted that the Km values of the native
polyamines (Put, Spd, Spm) were 208.2, 2.46, and 1.34 μM,
respectively, in L1210 cells.2d Thus, any of the three native
polyamines could theoretically be used as a rescue agent in
DFMO-treated cells because of the cell’s ability to interconvert
internal polyamine pools (see Figure 2). Putrescine transport
was easily blocked, however, because of its high Km value, and
spermine at high doses was toxic to cells.2d In this regard,
spermidine was more challenging to inhibit than putrescine,
was less toxic to cells than spermine, and was thus preferred as
the competing native polyamine in our system.
In summary, the disubstituted systems (5 and 8) and the
triamide 7b containing the longer polyamine sequence were all
found to be significantly more toxic than 6a and 6b. As
discovered in subsequent studies, the relative toxicities of these
compounds significantly influenced the doses that could be
used in combination with DFMO.
DFMO CHO Studies. Inhibition of ODC by DFMO can
lead to an increase in polyamine transport activity to maintain
intracellular polyamine homeostasis (see Figure 2). The high
A nontoxic PTI would be expected to inhibit Spd entry, and
the cell treated with DFMO+Spd+PTI would be expected to
resemble cells treated with DFMO only, i.e., with ∼50%
viability. With this assay in place, the effectiveness of each
potential PTI was tested and compared. In a 96-well plate
format, CHO cells were treated with an IC50 dose of DFMO
(4.2 mM), a fixed concentration of Spd (1 μM), and increasing
doses of the PTI compound (from 0 μM to its approximate
IC10 value). The cells were then incubated for 48 h at 37 °C.
Sample results are shown in Figure 3 for 6a and 6b. The
EC50 value is the concentration of the PTI compound needed
to reduce the cell viability halfway between the % viability
observed with DFMO+Spd control (green line in Figure 3) and
the % viability observed for the DFMO-only control (red line).
The EC50 values for 1b, 6a, 6b, and 7a were 1.37, 0.34, 0.42,
and 60 μM, respectively, and are listed in Table 1. In each case,
the EC50 value was well below the IC10 value of the compound
tested, and the innate toxicity of the PTI compound was,
therefore, excluded as a major contributing factor to the results
obtained.
Figure 2. Overview of polyamine metabolism and the combination
therapy of DFMO+PTI. ODC: ornithine decarboxylase, SOX:
spermine oxidase, PAO: polyamine oxidase, SSAT: spermidine-
spermine acetyl transferase. The combination therapy of DFMO
+PTI is expected to generate sustained intracellular polyamine
depletion, as both biosynthesis and import should be inhibited. In
the absence of a PTI, exogenous spermidine can enter and rescue
DFMO-treated cells. This rescue effect is due to biosynthetic and
catabolic processes which can interconvert each of the native
polyamines (e.g., spermidine, Spd) to each of the other native
polyamines as needed.
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Figure 3. Ability of (a) 6a (Trimer44) and (b) 6b (Trimer44NMe) to prevent Spd rescue of DFMO-treated CHO cells. Cells were incubated for 48
h at 37 °C with the respective conjugate, DFMO (4.2 mM), and Spd (1 μM). A 1 mM AG solution was incubated with cells for 24 h prior to drug
addition. Column 1 is the untreated CHO control, column 2 shows the % relative cell viability when dosed with PTI only at or above the
concentrations tested in the figure and shows that the PTI is nontoxic at the tested doses, column 3 is the Spd-only control at 1 μM, column 4 is the
DFMO-only control at 4.2 mM, and columns 5−7 have fixed concentrations of DFMO (4.2 mM) and Spd (1 μM) with increasing concentrations of
PTI as indicated in each lane. The PTIs at their IC10 values when tested alone gave ≥90% viability as determined in separate control experiments; the
respective IC10 values are listed in Table 1. Because of the high potency of 6a and 6b, they only needed to be dosed to 0.8 μM to illustrate their
respective dose dependence as a PTI agent. Note: this effective concentration was significantly below the IC10 value of both 6a and 6b (10 μM).
a e
−
Table 2. Biological Evaluation of Polyamine Derivatives (1b and 6−8) in L3.6pl Cells
d
compound
Ki (nM)
L3.6pl IC10 (μM)
EC50 in DFMO+Spd+PTI expt (μM)
L3.6pl IC50 (μM)
f
1b (L-Lys-Spm)
6a (Trimer44)
26
>100
10
1.06
0.77
1.40
89.8
NE
>100
36
>100
6b (Trimer44NMe)
7a (Triamide44)
7b (Triamide444)
7c (Triamide343)
8a (mBn44)
55
10
>100
398
ND
ND
ND
ND
>100
1
>100
9.7 ( 0.5)
47.5 ( 2.1)
52.2 ( 2.0)
7.5 ( 0.4)
8
NE
e
0.1
NE
e
8b (mBn444)
0.01
NE
a
b
L3.6pl cells were incubated with 250 μM AG for 24 h prior to drug addition. For experiments other than the Ki determinations, L3.6pl cells were
c
d
3
incubated for 48 h at 37 °C with the respective conjugate. All experiments were performed in triplicate. Ki values were determined via H-
spermidine uptake measurements taken over 15 min at 37 °C, and the Km of Spd in L3.6pl cells was determined to be 666 nM. 8a (0.1 μM) and 8b
(0.01 μM) displayed viability within 8% of the untreated control. ND = not determined; NE = not effective when dosed at its IC10 value. The L3.6pl
e
f
IC50 value for 1b was >500 μM. DFMO (8 mM) and Spd (1 μM) were used in the EC50 experiments.
As seen in Figure 3, compounds 6a and 6b effectively
blocked the import of Spd (1 μM) in a dose-dependent fashion.
For example, in Figure 3a, increasing levels of 6a provided
decreased viability in the presence of a fixed concentration of
DFMO and Spd. Direct comparisons of 1b, 6a, 6b, and 7a in
CHO cells (see Table 1) demonstrated that compounds 6a and
6b were excellent inhibitors of Spd import. Using the EC50
values of 1b, 6a, 6b, and 7a in CHO cells as listed in Table 1,
6a was 4-fold more potent than 1b and 176-fold more potent
than 7a in the CHO spermidine rescue assay.
The high toxicity of compounds 7b, 8a, and 8b was reflected
in their low IC10 values, which severely limited the amount of
these compounds that could be dosed to CHO cells with no
toxicity. In this regard, a toxic compound such as 8a, which
could only be dosed up to 0.01 μM (its IC10), was ineffective in
inhibiting the uptake of spermidine. This was not surprising
because the low IC10 value of 8a (0.01 μM) imparted a severe
disadvantage on a molar basis in terms of its ability to
outcompete Spd (1 μM) for the PTS. In CHO cells, 7b could
only be tested up to 1 μM, while 8a and 8b could only be
tested up to 0.01 μM. When tested up to their respective IC10
value in DFMO-treated CHO cells, none of these toxic
compounds (7b, 8a, and 8b) could effectively prevent the
rescue effect of the added Spd (1 μM).
In summary, the CHO experiments clearly demonstrated
that 6a (trimer44) and 6b (trimer44NMe) were more effective
than the other two PTIs: 1b (L-Lys-Spm) and triamide 7a.
L3.6pl Pancreatic Cancer Cell Line Studies. There is a
strong need for new medicines for pancreatic cancer, as the five
year survival rate for patients with metastatic pancreatic cancer
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Figure 4. Ability of (a) 1b (Lys-Spm), (b) 6a (Trimer44), (c) 6b (Trimer44-NMe), (d) 7a (Triamide44), and (e) 7c (Triamide343) to prevent Spd
rescue of DFMO-treated L3.6pl cells. Cells were incubated for 48 h at 37 °C with increasing doses of the respective PTI, in the presence of fixed
concentrations of DFMO (8 mM) and Spd (1 μM). A 250 μM solution of AG was incubated with cells for 24 h prior to compound addition.
Column 1 is the untreated L3.6pl control, and column 2 shows the % relative cell viability when dosed with the PTI alone at the highest
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Figure 4. continued
concentration tested in the figure and shows that the PTI is nontoxic. Note: the Spd-only control at 1 μM gave 100% viability (not shown), column
3 is the DFMO-only control at 8 mM, and columns 4−8 have fixed concentrations of DFMO (8 mM) and Spd (1 μM) with increasing
concentrations of PTI as indicated in each panel. The EC50 could not be determined even at the IC10 value of 7c (Figure 4e).
Figure 5. Intracellular polyamine levels in L3.6pl cells after 48 h incubation at 37 °C with DFMO (8 mM), Spermidine (Spd, 1 μM), or PTI 6b (7
μM) alone or in combination. Total PA = total polyamine levels (putrescine+spermidine+spermine) expressed in nmol/mg protein. Spd/Spm =
spermidine/spermine molar ratio. Conditions shown in red had appreciable growth reduction (DFMO only in lane 2 gave 64% growth vs untreated
control, and the combination of 6b+Spd+DFMO in lane 7 gave 70% growth). A 250 μM AG solution was incubated with cells for 24 h prior to
compound addition.
is just 6%.27 For this reason, the PTI panel was also evaluated in
the metastatic human pancreatic cancer cell line, L3.6pl. The
L3.6pl cell line28 was chosen because of its K-ras mutation,
which is prevalent in many human cancers and has been linked
to increased polyamine uptake.29 We were also interested in
whether this pancreatic cell line, which was optimized for its
high metastatic potential,28 was sensitive to the DFMO+PTI
combination therapy.
The IC50 values of the compounds in L3.6pl cells followed
the same trend as seen in CHO (see Tables 1 and 2). First, the
trisubstituted system 6a was significantly less toxic than the
disubstituted model 8a. Second, increased polyamine length
(7a vs 7b, and 8a vs 8b) again led to increased toxicity. As a
consistent trend in both CHO and L3.6pl cells, compounds 1b,
6a, 6b, and 7a were the least toxic PTIs tested (right column
Table 2, all had IC50 values in L3.6pl cells >100 μM).
7c in DFMO-treated L3.6pl cells. The low IC10 values of 7b (1
μM), 8a (0.1 μM), and 8b (0.01 μM) again limited their ability
to compete with Spd at 1 μM. As a result, when these toxic
compounds (7b, 8a, and 8b) were tested at their respective
IC10 values in L3.6pl cells, they were unsuccessful in inhibiting
the uptake of Spd (1 μM, data not shown). Even though the
IC50 values indicated that triamide 7c (L3.6pl IC50: 47.5 μM)
was 4.9-fold less toxic than 7b (9.7 μM), it was ineffective at its
IC10 value (8 μM) in inhibiting Spd (1 μM) uptake in DFMO-
treated L3.6pl cells Figure 4e). These collective results are
consistent with those found in the CHO experiments and
further illustrated the need for PTIs with low toxicity (high IC10
values) and high potency. Along these lines, we noted the
increased potency of 1b in L3.6pl cells and low toxicity of 1b in
both CHO and L3.6pl cells, both of which were desirable PTI
properties.
DFMO L3.6pl Studies. As performed previously with CHO
cells, the 48 h IC50 of DFMO (8 mM), the minimum
concentration of Spd needed to rescue DFMO-treated L3.6pl
cells back to their maximum possible viability (typically 1 μM
Spd gave 85−90% viability in L3.6pl cells), and the IC10 value
of each compound were determined in L3.6pl cells (Table 2).
Additionally, Ki values were determined to assess the relative
affinity of each compound for the PTS and were obtained via
uptake experiments over 15 min at 37 °C using 3H-spermidine
and the most promising PTIs: 1b, 6a, 6b, and 7a (Table 2).
Figure 4 illustrates the results obtained with 1b, 6a, 6b, 7a, and
Because of potency and dosing issues, the EC50 values could
only be determined for the compounds with IC10 values (≥10
μM) in L3.6pl cells. EC50 values for 1b, 6a, 6b, and 7a were
1.06 μM, 0.77 μM, 1.40 μM, and 89.8 μM, respectively. We
noted that compounds 1b, 6a, and 6b had low toxicity and were
very effective PTIs in the L3.6pl cell line with IC50 values >100
μM and L3.6pl EC50 values ≤1.4 μM. Compound 7a was also
nontoxic (IC50 >100 μM) but was significantly less potent as a
PTI (EC50 value = 89.8 μM). In this regard, compounds 1b, 6a,
and 6b had the optimal balance of low toxicity and low EC50
values. Rewardingly, the Ki values also tracked well with PTI
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Figure 6. Design comparisons between 6−8.
performance (and EC50 values) with 1b, 6a, and 6b having Ki
values (≤55 nM) significantly lower than that of 7a (398 nM,
Table 2). These findings supported the competitive inhibition
observed with these polyamine-containing compounds.
Intracellular Polyamine Levels. The effect of the
combination therapy of DFMO+6b on the intracellular
polyamine pools of L3.6pl cells was determined. As shown in
Figure 5, putrescine levels were virtually undetectable and
spermidine levels were dramatically reduced in each experiment
when the ODC inhibitor DFMO was used at its 48 h IC50
concentration (8 mM). In contrast, spermine levels were
maintained or increased in each experimental condition
evaluated. This is consistent with other studies of DFMO,
where cells tended to maintain or increase their spermine pools
when challenged with DFMO or DFMO in combination with a
PTI.4a,30,23 Compound 6b was treated at five times its EC50
concentration (EC50= 1.4 μM), which was below its IC10 value
(10 μM) and was, therefore, not toxic at the concentration used
(7 μM).
Several interesting trends were apparent. First, the PTI 6b
showed reductions in intracellular spermidine levels (27%
decrease) when dosed alone (Figure 5: lane 5) or in
combination with exogenous spermidine (33% decrease, lane
6). The combination therapy of DFMO+PTI 6b in the
presence of a potential rescuing dose of spermidine (1 μM)
revealed sharp reductions in putrescine (undetectable) and
spermidine levels (87% decrease vs control) and a 67% increase
in spermine levels (lane 7). These results are consistent with
the effect of other PTIs on intracellular polyamine levels.23
Second, an unexpected result was that the DFMO+Spd
control (Figure 5: lane 4, 103% relative growth, where the
untreated control is set to 100%) had similar intracellular
polyamine levels as the combination of 6b+Spd+DFMO (lane
7, 70% relative growth) and DFMO-only control (lane 2, 64%
relative growth). Closer examination revealed that the primary
difference between these experiments was the measured
intracellular spermidine levels (Figure 5). As intracellular
polyamine levels decreased, decreasing intracellular Spd/Spm
ratios were observed. Spermidine is important for growth and
provides a means to maintain cellular spermine content via
biosynthesis (Figure 2).31 L3.6pl cells were observed to have
normal growth when the Spd/Spm ratio ranged from 2.86 to
0.37. Our data suggest, however, that when the Spd/Spm ratio
and Put+Spd+Spm levels drop below a critical threshold, cell
growth is reduced. These observations are consistent with other
published accounts where critical levels of polyamines must be
maintained for proper cell function and growth.4a,31
In short, the reductions observed in both cell growth and
intracellular polyamine levels when 6b is dosed in combination
with DFMO+ Spd are all consistent with 6b acting as an
effective PTI. The low Ki value of 6b (55 nM) observed in
competition experiments with 3H-Spd also support this
conclusion.
A summary of the design comparisons of the aryl-polyamine
series 6−8 is illustrated in Figure 6. First, introduction of
terminal N-methyl groups was found to decrease the sensitivity
of the compound to amine oxidases, without significant loss of
its potency as a PTI. In terms of PTI performance, compound
6a (Trimer44) had consistent potency in both the CHO (EC50
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value = 0.34 μM) and L3.6pl (EC50 value = 0.77 μM) cell lines.
The N-methyl derivative 6b also displayed similar potency in
CHO (EC50 value = 0.42 μM) and L3.6pl cells (EC50 = 1.4
μM). While the potencies were similar, the performance of
these two compounds in the absence of aminoguanidine was
different. It is important to note that all of the spermidine
rescue experiments were performed in the presence of
aminoguanidine (AG), a known inhibitor of copper-dependent
oxidases which cause the terminal deamination of polyamines.32
Indeed, AG was required to maintain the stability of both
spermidine and the polyamine-based PTI under evaluation
because compounds containing free-terminal primary amines
were potential substrates for amine oxidases in the fetal bovine
serum used for cell culture.33
One method of evaluating the sensitivity of polyamine-based
compounds to amine oxidases is to evaluate their innate toxicity
in the presence and absence of AG. If the compound is not a
substrate for amine oxidases, then it should retain its structure
and IC50 value in the presence and absence of a nontoxic dose
of AG.33 The L3.6pl IC50 values of 6a and 6b in the presence of
AG (250 μM) were 102.8 ( 4.4) and 179.0 ( 11.7) μM,
respectively. The L3.6pl IC50 values of 6a and 6b in the absence
of AG (250 μM) were 188.3 ( 7.6) and 183.4 ( 10.5) μM,
respectively. These data support the contention that the N-
methyl derivative 6b retained its potency, whereas for 6a, a
good fraction of its activity was lost over the 48 h incubation
period. These findings were consistent with observations made
with other N-methyl polyamine derivatives.33 In this regard, 6b
provided an alternative design with decreased sensitivity to
amine oxidases and with similar potency as 6a. Future studies
are needed, however, to determine if this property provides the
intended advantage in vivo. Indeed, decreased sensitivity to
amine oxidases could be critical because in vivo experiments are
performed without AG present.
Second, a comparison of 6a and 7b provided insight into the
role of the tether connecting the homospermidine motif to the
aryl ring core. Because 7b contains an amide link, the nitrogen
closest to the ring does not bear a formal positive charge at
physiological pH. In this regard, 7b represents an extended
version of 6a, where the homospermidine messages (Figure 6)
are presented further away from the aryl core due to the six
atom linker in 7b (i.e., CONH(CH2)4). This modification from
short tether to longer tether introduced significant toxicity
(L3.6pl IC50 of 7b = 9.7 μM), which resulted in a dramatically
lower IC10 value for 7b (1 μM) and poor PTI properties
compared to 6a. In summary, short tethers provided less toxic
PTIs with greater potency.
Third, a comparison of 7a, 7b, and 7c revealed that 7a was
significantly less toxic than 7b and 7c, which resulted in IC10
values in L3.6pl cells of >100, 1, and 8 μM for 7a−c,
respectively (Table 2). As shown in Table 2, the high toxicities
observed with 7b and 7c (as well as 8a and 8b) precluded them
from being effective PTI agents. While 7a could act as a PTI, its
CHO and L3.pl EC50 values were 60 and 89.8 μM, respectively.
These values were significantly higher than the EC50 values
obtained with 6a and 6b, indicating that at least in these cell-
based assays 7a had lower potency. We speculate that this was
due to the shorter polyamine message (putrescine) displayed
by 7a versus the homospermidine message presented by 6a and
6b (Figure 6).
PTS.2d Indeed, it is well established that affinity increases across
the polyamine series: diamines < triamines < tetraamines.2d
This insight could also be applied to 6a and 7a. Even though
both systems have six atoms separating the aryl core from the
second nitrogen of the polyamine chain [i.e., 6a: CH2NH-
(CH2)4 vs 7a: CONH(CH2)4], the presence of the charged
nitrogen closest to the ring in 6a presented a higher affinity
homospermidine motif instead of the putrescine message
presented by amide 7a (Figure 6). This is consistent with both
the significantly lower Ki value observed with 6a (Table 2) and
the extensive SAR studies by our group with other aryl-
polyamine systems.13a
CONCLUSIONS
■
These studies indicated that a platform which displayed three
homospermidine messages provided the most potent PTI of
the series evaluated. The triamides (7b and 7c), meta-
substituted analogues (8a and 8b), and the para-substituted
5a system had significant toxicity in the cell lines examined
which limited their application as PTIs. The third polyamine
arm was shown to be a key part of the PTI design and gave rise
to promising compounds (6a and 6b), which efficiently blocked
the import of spermidine (1 μM) into DFMO-treated cells as
3
well as the import of H-spermidine into untreated cells. PTI
6b when dosed with DFMO and Spd produced dramatic
reductions in intracellular polyamine levels vs the untreated
control as well as a 30% reduction in L3.6pl growth after 48 h
incubation.
Ultimately, compound 6a proved to be an excellent PTI with
high potency as evidenced by its submicromolar EC50 values in
both cell lines evaluated in vitro. The fact that the N-methyl
analogue 6b was shown to have decreased sensitivity to amine
oxidases and lower toxicity than 6a provided a new compound
with desirable properties for in vivo evaluations. A recent patent
has also demonstrated inhibition of putrescine uptake by 6a in
DFMO-treated L3.6pl cells as well as colon cancer (SW620)
and other pancreatic cancer cell lines (MiaPaca-2).26 Additional
experiments with 6a also demonstrated its efficacy in
combination with DFMO in the presence of exogenous
putrescine or spermine in L3.6pl cells (see Supporting
Information). Future experiments will test whether these in
vitro trends with 6a and 6b translate to the appropriate in vivo
models.6
Lastly, the development of PTIs provides unique tools to
examine biological processes dependent upon polyamine
transport. For example, the triamide PTI 7a, which showed
modest potency in blocking Spd uptake in this report, was
recently shown to be an efficient inhibitor of putrescine uptake
in bacteria.34 Specifically, 7a was used to understand the
interplay between the putrescine uptake transporter PlaP and
the swarming behavior of Proteus mirabilis, a Gram-negative
bacterium known to cause urinary tract infections in humans.34
In this regard, compounds with PTI properties may find novel
applications in both mammalian and bacterial systems.
EXPERIMENTAL SECTION
■
Materials. 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. All reactions
were carried out under an N2 atmosphere. H and 13C spectra were
1
In direct comparisons of putrescine and homospermidine in
L1210 cells, the Km of putrescine was significantly higher than
that of homospermidine, indicating a lower affinity for the
recorded at 400 and 75 MHz, respectively. TLC solvent systems were
listed as volume percents, and NH4OH referred to concentrated
aqueous ammonium hydroxide. All tested compounds provided
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3
satisfactory elemental analyses as proof of purity (≥95%). These are
provided in the Supporting Information.
3H-Spd and L is the concentration of H-Spd used in the assay (1
μM). The Km for Spd (666 nM) was calculated by using solutions of
3H-Spd ranging in concentration from 0.3 to 3 μM and plotting the
Biological Studies. CHO, CHO-MG*, and L3.6pl cells were
grown in RPMI 1640 medium supplemented with 10% fetal bovine
serum (FBS) and 1% penicillin/streptomycin. Note: the media must
contain L-proline (2 μg/mL) for proper growth of the CHO-MG*
cells. All cells were grown at 37 °C under a humidified 5% CO2
atmosphere. Aminoguanidine (1 mM for CHO and CHO-MG*, and
250 μM for L3.6pl) was added to the growth medium to prevent
oxidation of the compounds by the enzyme (bovine serum amine
oxidase) present in calf serum. Cells in early to mid log phase were
used.
IC50 Determinations. Cell growth was assayed in sterile 96-well
microtiter plates (Costar 3599, Corning, NY). CHO and CHO-MG*
cells were plated at 10 000 cells/mL. L3.6pl cells were plated at 5000
cells/mL. Drug solutions (10 μL per well) of appropriate
concentration in phosphate-buffered saline (PBS) were added after
an overnight incubation for each CHO cell line (90 μL of cell
suspension used). After exposure to the drug for 48 h, cell growth was
determined by measuring formazan formation from the 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-2H-
tetrazolium, inner salt (MTS) using a SynergyMx Biotek microplate
reader for absorbance (490 nm) measurements.35 All experiments
were run in triplicate.
inverse of [3H-Spd] versus the inverse of the pmol Spd/μg protein/
min . The Km = −1/x intercept.
(S)-2,6-Diamino-N-(3-((4-((3-aminopropyl)amino)butyl)-
amino)propyl)hexanamide (1b, L-Lys-Spm). Compound 1b was
synthesized by a modified method of Weeks et al.23 1H NMR (D2O) δ
3.92 (t, 1H), 3.38 (m, 1H), 3.27 (m, 1H), 3.12 (m, 10H), 2.91 (t, 2H),
2.18 (m, 2H, 1.89 (m, 4H), 1.77 (m, 4H), 1.71 (m, 2H), 1.39 (m,
2H); matched literature values.15,23 HRMS (FAB) m/z calcd for
C16H43Cl5N6O (M + H)+ 331.3180, found 331.3175. Anal.
C16H43Cl5N6O·H2O, CHN.
N1,N1′,N1″-(Benzene-1,3,5-triyltris(methylene))tris(N4-(4-
aminobutyl)butane-1,4-diamine) (6a). The N-H Boc-protected
compound 20a (0.3948 g, 0.331 mmol) was dissolved in absolute
ethanol (10 mL) and stirred at 0 °C for 10 min. A 4 N HCl solution
(20 mL) was added to the reaction mixture and stirred overnight. The
solution was concentrated, giving the respective amine HCl salt 6a
(0.216 g, 71%). 1H NMR (D2O, 400 MHz): δ 7.69 (s, 3H, aromatic),
4.35 (s, 6H, CH2), 3.20 (t, 6H, CH2), 3.06 (m, 18H, CH2), 1.79 (m,
24H, CH2). 13C NMR (D2O): δ 187.9, 135.7, 135.2, 53.2, 49.8, 49.7,
41.7, 26.8, 25.7, 25.6. Matched the literature spectrum, as 6a was
synthesized previously.13b Anal. C33H78N9Cl9·0.2H2O, CHN
N1,N1′,N1″-(Benzene-1,3,5-triyltris(methylene))tris(N4-(4-
(methylamino)butyl)butane-1,4-diamine) (6b). The N-Me Boc-
protected compound 20b (0.23 g, 0.17 mmol) dissolved in absolute
ethanol (10 mL) was stirred at 0 °C for 10 min. A 4 N HCl solution
(20 mL) was added to the reaction mixture and stirred overnight. The
solution was concentrated, giving the respective amine HCl salt 6b
(0.151 g, 85%). 1H NMR (D2O, 400 MHz): δ 7.69 (s, 3H, aromatic),
4.35 (s, 6H, CH2), 3.20 (t, 6H, CH2), 3.06 (m, 18H, CH2), 2.74 (s,
9H, CH3), 1.80 (m, 24H, CH2). 13C NMR (D2O): δ 135.7, 135.2,
53.2, 51.1, 49.73, 49.69, 35.6, 25.7, 25.5. HRMS (FAB) m/z calcd for
C36H76N9 (M + H)+ 634.6218, found 634.6197. Anal. C36H84N9Cl9 ·
1.1H2O, CHN.
HPLC and Polyamine Level Determination.36 Briefly, L3.6pl
cells (450 000 cells/6 mL media) were incubated with aminoguanidine
(250 μM) at 37 °C for 24 h, each compound was added either alone or
in combination with other agents as indicated in Figure 5, and the cells
were incubated for another 48 h at 37 °C. The cells were then washed
with PBS and lysed using a 0.2 M perchloric acid/1 M NaCl solution
(200 μL), sonicated, and centrifuged, and the resultant supernatant
and pellet were separated. The standard 1,7-diaminoheptane (30 μL of
1.5 × 10−4 M) was added to the polyamine-containing supernatant
(100 μL) and then treated at 65 °C for 1 h with 1 M Na2CO3 (200
μL) and 18.5 mM dansyl chloride (400 μL) to generate the respective
N-dansylated polyamines. Proline (1 M, 100 μL) was added, and the
reaction mixture was shaken on a rotary shaker for 20 min at 65 °C.
The dansylated products were then extracted into chloroform (2 mL),
the organic layer was separated and concentrated, the residue was
redissolved in MeOH (1 mL), and each polyamine was then quantified
via HPLC analysis.36 The protein content of the pellet was quantified
using the Pierce BCA Protein assay kit from Thermo Scientific. Final
results are expressed as nmol polyamine/mg protein. Investigation into
the media used for these experiments (which contained 10% FBS)
revealed a total of 2.6 nmol of putrescine, no detectable spermidine,
and 1.6 nmol of spermine that were present in the 6 mL of media used
for the experiments. In this regard, minimal exogenous polyamines
were available to the growing L3.6pl cells in the nonspermidine-spiked
experiments.
N1,N3,N5-Tris(4-((4-aminobutyl)amino)butyl)benzene-1,3,5-
tricarboxamide (7a). A solution of Boc-protected 11a (910 mg, 0.67
mmol) was dissolved in absolute ethanol (70 mL) and stirred at 0 °C
for 10 min. A 4 M HCl solution (40 mL, 160 mmol) was added to the
reaction mixture dropwise and stirred at 0 °C for 30 min and then at
room temperature overnight. The solution was concentrated in vacuo
to give 7a as a white solid (625 mg, 99%); 1H NMR (D2O) δ 8.28 (s,
3H), 3.45 (t, 6 H), 3.14 (q, 12H), 3.07 (t, 6H), 1.78 (m, 24H); 13C
NMR (D2O) δ 172.0, 138.0, 131.8, 50.3, 49.9, 42.4, 41.8, 28.6, 27.0,
26.1, 25.8. HRMS (FAB) m/z calcd for C33H63N9O3 (M + H)+
634.5053, found 634.5129. Anal. C33H69Cl6N9O3·3.5H2O: CHN
N1,N3,N5-Tris(4-((4-((4-aminobutyl)amino)butyl)amino)-
butyl)benzene-1,3,5-tricarboxamide Nonahydrochloride (7b).
A method similar to that described above for 7a was used to make 7b
using Boc-protected amine 11b. Light pink solid, 84%. 1H NMR
(D2O) δ 8.29 (s, 3H), 3.46 (t, 6H), 3.15 (24 H), 3.05 (t, 6H), 1.79 (m,
36H); 13C NMR (D2O) δ171.8, 137.8, 131.9, 50.6, 49.6, 42.4, 41.5,
27.1, 28.8, 27.1, 26.8, 25.8. HRMS (FAB) m/z calcd for C45H91N12O3
(M + H)+ 847.7337, found 847.7332. Anal. C45H99Cl9N12O3·6H2O,
CHN
Ki Determinations. The inhibition constant for selected PTIs were
determined following the protocol of Weeks et al.23 Briefly, L3.6 pl
cells (100 K/well) were seeded into a 24-well plate for log phase
growth and incubated with 5% CO2 for 24 h at 37 °C. The media was
then changed with preheated Hanks Balanced Salt Solution (HBSS,
containing Ca2+ and Mg2+) at 37 °C. 3H-Spd (Perkin-Elmer Inc.,
Boston, MA, fixed at 1 μM) was added with the respective PTI at
different PTI concentrations (0, 0.1, 0.3, 1, 2, 3 μM). Cells were
incubated at 37 °C for 15 min. The cells were then washed with cold
HBSS and lysed with 0.1% sodium dodecyl sulfate (SDS) in water
(500 μL). Each cell lysate was then transferred to an Eppendorf tube
and centrifuged at 15 000 rpm for 15 min. A sample of each
supernatant (200 μL) was transferred into a scintillation vial
containing 2 mL of Scintiverse BD, and the resulting scintillation
counts were measured. The amount of protein was determined using
the Pierce BCA protein assay kit from the remaining lysate volume
(approximately 300 μL) to normalize the radioactive counts obtained
N1,N3,N5-Tris(3-((4-((3-aminopropyl)amino)butyl)amino)-
propyl)benzene-1,3,5-tricarboxamide Nonahydrochloride (7c).
The respective solution of the Boc-protected compound 11c (0.40 g,
0.24 mmol), dissolved in absolute ethanol (25 mL), was stirred at 0 °C
for 10 min. A 4 N HCl solution (15 mL, 60 mmol) was added
dropwise to the reaction mixture and stirred at 0 °C for 20 min and
then at room temperature overnight. The solution was concentrated in
vacuo to give the respective amine HCl salt as a solid 7c (0.21 g, 80%
1
yield). H NMR (D2O): δ 8.35 (s, 3H, aromatic), 3.56 (t, 6H, CH2),
3
3.23−3.08 (m, 30H, CH2), 2.17−2.02 (m 12H), 1.89−1.75 (br s,
12H). 13C NMR (D2O): δ 172.1, 137.6, 132.0, 50.0, 49.9, 48.3, 47.5,
39.9, 39.5, 28.6, 26.7, 25.8, 25.7. Anal. C39H87N12O3Cl9·3.44H2O,
CHN.
(pmol H-Spd/μg protein). Ki and Km values were determined using
double reciprocal Lineweaver-Burk plots. The Ki value was determined
from the equation Ki = IC50/(1 + (L + Km)), where IC50 is the
concentration of PTI required to block 50% of the relative uptake of
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N1,N1′-(1,3-Phenylenebis(methylene))bis(N4-(4-aminobutyl)-
butane-1,4-diamine) (8a). Compound 15a (174 mg, 0.21 mmol)
was dissolved in EtOH (30 mL) and stirred at 0 °C for 10 min, and
then a 4 M HCl solution (12 mL, 48 mmol) was then added dropwise
and stirred for 30 min. The temperature was then allowed to rise to
room temperature, and the solution was then stirred under N2 for 4 h.
The solvents were then removed in vacuo to give 8a as a white solid
(80 mg, 95% yield). 1H NMR (D2O) δ 7.57 (s, 3H), 7.55 (s, 1H), 4.26
(s, 4H), 3.20−3.01 (m, 16H), 1.76 (m, 16H); 13C NMR (D2O) δ
134.7, 133.9, 133.8, 133.0, 53.6, 49.8, 49.5, 41.7, 26.8, 25.7. HRMS
(FAB) m/z calcd for C24H48N6 (M + H)+ 421.3940, found 421.4013.
Anal. C24H54Cl6N6·0.6H2O, CHN.
removed in vacuo, and the residue was redissolved in CH2Cl2. The
organic layer was then washed with 0.1 M HCl (24 mL), followed by
deionized H2O (24 mL) and saturated aq Na2CO3 (24 mL). The
organic layer was then separated, dried over anhydrous Na2SO4,
filtered, and concentrated to give a crude solid which was then purified
by flash column chromatography to afford 11a (1.05 g, 56%) as a
white solid (Rf = 0.25; 4% MeOH/96% CH2Cl2); 1H NMR (CDCl3) δ
8.41 (s, 3H), 4.75 (br s, 3H), 3.49 (m, 6H), 3.23−3.06 (m, 18H),
2.78−1.30 (m, 78H); 13C NMR (CDCl3) δ 166.5, 156.1, 155.7, 135.2,
128.5, 79.3, 78.9, 46.6, 40.1, 39.8, 28.4, 27.3, 26.4. HRMS (FAB) m/z
calcd for C63H111N9O15 (M + Na)+ 1256.8092, found 1256.8105. Anal.
C63H111N9O15·H2O, CHN.
N1,N1′-(1,3-Phenylenebis(methylene))bis(N4-(4-((4-
aminobutyl)amino)butyl)butane-1,4-diamine) (8b). Compound
15b (130 mg, 0.11 mmol) was dissolved in EtOH (10 mL) and stirred
at 0 °C for 10 min, and a 4 M HCl solution (6 mL, 24 mmol) was
added dropwise and stirred for 30 min. The temperature was then
allowed to rise to room temperature, and the solution was stirred
under N2 for 4 h. The solvents were then removed in vacuo to give a
white solid (95 mg, 98%). 1H NMR (D2O) δ 7.59−7.57 (m, 4H), 4.30
(s, 4H), 3.17−3.04 (m, 24H), 1.79−1.77 (m, 24H); 13C NMR 187.9,
134.4, 133.8, 133.7, 132.9, 53.4, 49.6, 49.3, 41.5, 26.6, 25.5. HRMS
(FAB) m/z calcd for C32H66N8 (M + H)+ 563.5410, found 563.5483.
Anal. C32H74Cl8N8·2.5H2O, CHN.
[4-(3,5-Bis(4-[tert-butoxycarbonyl-(4-tert-butoxycarbonyl)-
(4-tert-butoxycarbonylaminobutyl)amino]butylcarbamoyl)-
benzoylamino)butyl](4-tert-butoxycarbonylaminobutyl)-
carbamic Acid tert-Butyl Ester (11b). A similar procedure was used
for 11b, except that Boc-protected polyamine 10b16 was used. White
1
solid, 75%. H NMR (CDCl3) δ 8.19 (s, 3H), 3.42 (t, 6H), 3.17 (t,
30H), 1.71−1.41 (m, 117H); 13C NMR (CDCl3) δ 166.4, 156.2,
155.7, 135.4, 128.9, 79.5, 46.8, 45.8, 41.2, 40.5, 39.5, 29.1, 28.5, 27.6,
26.5. Anal. C90H162N12O21, CHN.
Boc-Protected Triamide 11c. Tri-Boc spermine 10c (2.27 mmol,
1.14 g) was dissolved in CH2Cl2 (10 mL), and a solution of K2CO3
(2.27 mmol, 0.313 g) and water (6 mL) was added. Aliquat (1 drop), a
phase transfer catalyst, was added, and the mixture was stirred and
cooled to 0 °C. A solution of acid chloride 9 (0.69 mmol, 0.183 g) in
CH2Cl2 (10 mL) was added to the original mixture and after the
addition was complete was allowed to warm to rt. The reaction was
stirred vigorously overnight. After the reaction was complete, the
organic layer was washed with water (20 mL), separated, dried over
anhydrous Na2SO4, filtered, and concentrated. Column chromatog-
raphy (95% CH2Cl2/5% MeOH) was performed (Rf = 0.3). A second
column (80% ethyl acetate/20% CHCl3) was used to remove any
remaining impurities (Rf = 0.23). The final product was the oil 11c
Polyamines 10a, 10b, and 10c have all been previously synthesized
and characterized.16,17b,37,
tert-Butyl (3-Aminopropyl)(4-((tert-butoxycarbonyl)(3-((tert-
butoxycarbonyl)amino)propyl)amino)butyl)carbamate (Tri-
boc-Spermine 10c). As shown in Scheme 2, spermine free base
(1.0 g: 5.1 mmol) was dissolved in 25% MeOH/CH2Cl2 solution (200
mL) and stirred at rt, and 2-hydroxybenzaldehyde (0.63 g: 5.1 mmol)
was added dropwise. Anhydrous Na2SO4 (5.7 g: 40.2 mmol) was
added, and the reaction was stirred overnight at rt. 1H NMR (CDCl3)
showed complete conversion to 12 after overnight stirring at rt. Imine
1
1
12 was then consumed in the next step without purification. 12: H
(0.45 g, 40% yield). H NMR (CDCl3) δ 8.45 (br s, 3H), 7.93 (br s,
NMR (CDCl3): δ 8.35 (br s, 1H, CH), 7.35−7.19 (br s, 1H,
aromatic), 6.99−6.91 (br s, 1H aromatic), 6.90−6.81 (br s, 1H,
aromatic), 3.66 (br s, 2H, CH2), 2.80−2.56 (m, 10H, CH2). The
reaction mixture of 12 was cooled to 0 °C, and di-tert-butyl
dicarbonate (3.4 g, 15.6 mmol) was added as a solid. The reaction
was then stirred for 1.5 h at rt. Upon completion, the volatiles were
removed under reduced pressure to provide the tri-Boc imine 13,
which was immediately consumed in the next step without
purification. 13: 1H (CDCl3): δ 8.35 (s, 1H, CH), 3.59 (m, 2H,
CH2), 3.36−3.04 (m, 10H, CH2). The imine 13 was cleaved using
CH3ONH2 (1.60 g, 18.7 mmol) and Na2CO3 (2.0 g, 18.7 mmol).
(Note: Na2CO3 was slightly insoluble in MeOH). The reaction turned
a cloudy white and then was stirred for 2 h. The volatiles were
removed under reduced pressure and redissolved in CH2Cl2 (50 mL).
The organic layer was washed with aq Na2CO3 (∼10 wt %), and the
organic layer was separated, filtered, and concentrated (4.0 g). Column
chromatography (75% CH2Cl2/25% hexane) was used to elute the
oxime (Rf = 0.4) away from the desired product, which remained on
the column. The solvent system was then changed to 1% ammonium
hydroxide/5% methanol in dichloromethane to elute the product from
the column (Rf = 0.3). The product, 10c was a sticky oily liquid (1.14
g, 45%). 1H NMR (CDCl3) δ: 7.27 (s, 1H, NH), 3.37−3.05 (m, 10H,
CH2) 1.80−1.36 (m, 38H).
1.7H), 7.26 (br s, 0.46H), 5.48 (br s, 1.9H), 5.07 (br s, 0.9H), 3.51−
3.0 (m, 42H), 1.93−1.35 (m, 117H); 13C NMR (125 MHz, CDCl3) δ
165.9, 156.5, 156.0, 155.5, 135.5, 128.3, 79.9, 79.6, 78.9, 46.8, 46.2,
44.3, 43.9, 37.5, 36.5, 29.0, 28.6, 28.5, 28.3, 27.7, 26.1. Note: both the
1H and 13C NMR spectra gave rise to broad signals which were
expected for this compound type in CDCl3. Anal. C84H150N12O21·
0.9H2O, CHN.
Di-tert-butyl ((1,3-Phenylenebis(methylene))bis(azanediyl))-
bis(butane-4,1-diyl))bis((4-((tert-butoxycarbonyl)amino)butyl)-
carbamate (15a). To maximize the yield, MeOH was dried and
distilled prior to this reaction. A solution of 1,3-benzene dicarbox-
aldehyde 14 (50.0 mg, 0.37 mmol) in 25% MeOH/CH2Cl2 (10 mL)
was added to a stirred solution of Boc-protected amine 10a (453 mg,
1.26 mmol) in 25% MeOH/CH2Cl2 (15 mL). The reaction was then
stirred at room temperature under N2 overnight. After imine formation
was complete (by 1H NMR), the solvents were removed in vacuo and
the residue was redissolved in 50% MeOH/CH2Cl2 (20 mL). The
solution was then cooled to 0 °C followed by addition of predried
NaBH4 (85.3 mg, 2.26 mmol) in small portions, and the mixture was
stirred at room temperature under N2 for 2 h. The volatiles were then
removed under reduced pressure, and the residue was redissolved in
CH2Cl2 and washed three times with aqueous Na2CO3. The organic
layer was separated, dried over Na2SO4, filtered, and concentrated.
Column chromatography (Rf = 0.25, 25% hexanes/0.1% NH4OH/
74.9% distilled THF) provided compound 15a as a yellow oil (150 mg,
[ 4 - ( 3 , 5 - B i s ( 4 - [ t e r t - b u t o x y c a r b o n y l - ( 4 - t e r t -
butoxycarbonylaminobutyl)amino]butylcarbamoyl)-
benzoylamino)butyl](4-tert-butoxycarbonylaminobutyl)-
carbamic Acid tert-Butyl Ester (11a). A solution of Boc-protected
amine 10a16 (1.80 g, 5.01 mmol) and CH2Cl2 (12 mL) was prepared.
A solution of K2CO3 (1.65 g, 11.94 mmol) and water (20 mL) was
added to the original mixture. The tetraalkylammonium phase transfer
catalyst Aliquat (0.10 mL) was added and the mixture was cooled to 0
°C. 1,3,5-Benzenetricarboxylic acid chloride 9 (400 mg, 1.51 mmol) in
CH2Cl2 (24 mL) was added dropwise to the original mixture, allowed
to gradually warm, and stirred vigorously at room temperature for 6 h.
1
47%). H NMR (CDCl3) δ 7.28−7.19 (m, 4H), 3.77 (s, 4H), 3.14 (t,
12H), 2.65 (t, 4H), 1.55−1.42 (m, 52H); 13C NMR (CDCl3) δ 156.0,
155.6, 140.5, 128.5, 127.9, 126.7, 79.2, 79.1, 54.0, 49.3, 47.0, 46.9, 46.7,
46.5,40.2, 28.5, 28.4, 28.3, 27.4, 26.6, 26.1, 26.0, 25.6. HRMS for
C44H81N6O8 (M + H) calcd: 821.6110; found: 821.6103, Anal.
C44H80N6O8·0.12H2O, CHN.
Di-tert-butyl ((1,3-Phenylenebis(methylene))bis(azanediyl))-
bis(butane-4,1-diyl))bis((4-((tert-butoxycarbonyl)(4-((tert-
butoxycarbonyl)amino)butyl)amino)butyl)carbamate (15b).
To a stirred solution of Boc-protected amine 10b (705 mg, 1.33
1
After the reaction was complete (by H NMR), the solvents were
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Journal of Medicinal Chemistry
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mmol) in 25% MeOH/CH2Cl2 (20 mL) was added a solution of 1,3-
benzene dicarboxaldehyde 14 (52.3 mg, 0.39 mmol) in 25% MeOH/
CH2Cl2 (15 mL). The reaction was then stirred at room temperature
under N2 overnight. After imine formation was complete by 1H NMR,
the solvents were removed in vacuo, and the residue was redissolved in
50% MeOH/CH2Cl2 (25 mL). The solution was then cooled to 0 °C
followed by addition of NaBH4 (88.5 mg, 2.34 mmol) in small
portions, and the mixture was stirred at room temperature under N2
for 2 h. The solvents were then removed under reduced pressure, and
the residue was redissolved in CH2Cl2 and washed three times with
aqueous Na2CO3. The organic layer was separated, dried over Na2SO4,
filtered, and concentrated in vacuo. Column chromatography (Rf =
0.31 (10% MeOH/1% NH4OH/89% CH2Cl2)) provided compound
15b as a colorless oil (240 mg, 53%). 1H NMR (CDCl3) δ 7.32−7.19
(m, 4H), 3.77 (s, 4H), 3.15 (t, 20H), 2.65 (t, 4H), 1.60−1.44 (m,
78H); 13C NMR (CDCl3) δ 156.0, 155.5, 140.5, 128.4, 127.8, 126.7,
79.1, 54.0, 49.3, 46.9, 46.6, 40.2, 28.5, 27.4, 26.1. HRMS (FAB) m/z
calcd for C62H114N8O12 (M + H)+ 1163.8629, found 1163.8629. Anal.
C62H114N8O12, CHN.
CH2), 2.65 (t, 6H, CH2), 1.61−1.5 (m, 78H, CH2, CH3). 13C NMR
(D2O) δ 172.0, 138.0, 131.8, 50.3, 49.9, 42.4, 41.8, 28.6, 27.0, 26.1,
25.8.13b
Tri-tert-butyl (((((Benzene-1,3,5-triyltris(methylene))tris-
(azanediyl))tris(butane-4,1-diyl))tris((tert-butoxycarbonyl)-
azanediyl))tris(butane-4,1-diyl))tris(methylcarbamate) 20b. N-
Methylamine derivative 19 (0.72 g, 1.94 mmol) was dissolved in 25%
MeOH/CH2Cl2 (10 mL) and 1,3,5-triformylbenzene 18 (0.081 g, 0.50
mmol) was added. The reaction mixture was stirred overnight under a
N2 atmosphere. Loss of the starting material was monitored by loss of
1
the aldehyde CH (δ 10.21) in the H NMR. Upon the conversion of
the starting material, the solvent was removed by concentrating the
sample on a rotary evaporator. The crude material was redissolved in a
solution of 50% MeOH/CH2Cl2. To this new solution, NaBH4 (0.18
g, 4.77 mmol) was added at 0 °C and the reaction was stirred
overnight at rt under a N2 atmosphere. Loss of the intermediate imine
was monitored by 1H NMR. The solvent was removed under reduced
pressure. A base workup was performed by redissolving the residue in
CH2Cl2 and washing the organic layer with aqueous Na2CO3. The
organic layer was separated, dried over anhydrous Na2SO4, filtered,
and concentrated to give a crude oil (0.73 g). Column chromatog-
raphy (5% MeOH in CH3CN containing 1% NH4OH) was used to
reisolate the unreacted amine starting material (Rf = 0.65). The
column was then flushed with (50% MeOH in CH3CN containing 1%
NH4OH) to provide the pure trisubstituted aryl compound 20b (0.32
g, 39%). 1H NMR (CDCl3, 400 MHz): δ 7.15 (s, 3H, aromatic), 3.76
(s, 6H, CH2), 3.48 (m, 3H, NH), 3.19 (m, 18H CH2), 2.83 (s, 9H,
CH3), 2.65 (t, 6H, CH2), 1.75−1.40 (m, 78H CH2, CH3). 13C NMR
(CDCl3 100 MHz): 155.8, 155.6, 140.1, 126.7, 79.2, 54.0, 49.4, 48.7,
48.1, 46.9, 34.1, 28.5, 27.4, 26.6, 26.1, 25.3. HRMS (FAB) m/z calcd
for C66H124N9O12 (M + H)+ =1234.9364, found 1234.9335.
C66H123N9O12·0.8 H2O, CHN.
Benzene-1,3,5-triyltrimethanol (17). Trimethyl 1,3,5-benzene-
tricarboxylate 16 (5 g, 0.02 mmol) in dry THF (75 mL) was added
through a pressure-equalized addition funnel into a 500 mL round-
bottom flask containing LiAlH4 (3.8 g, 100 mmol) at 0 °C. The
mixture was allowed to warm to room temperature and stirred
overnight (14 h). The reaction was quenched by the slow addition of a
1:1 mixture of Celite and NaHSO4. The suspension was filtered, and
Celite was washed with EtOH (100 mL). The solvent was removed
under reduced pressure, and the crude triol 17 was obtained (4.71 g).
This crude solid was consumed in the next step without further
1
purification. H NMR (DMSO): δ 7.12 (s, 3H, aromatic), 5.12 (br s,
3H, CH2), 4.47 (s, 6H, OH), which matched its literature spectrum.13b
Benzene-1,3,5-tricarbaldehyde (18). The crude triol 17 (1 g,
5.9 mmol) was dissolved in 150 mL of tert-butyl alcohol. 2-
Iodoxybenzoic acid (IBX, 9.83g, 47.2 mmol) was added to the triol
mixture, and the reaction was stirred and heated to reflux in an oil bath
at 95 °C for 2 h. The reaction was monitored by TLC (35% EtOAc/
65% hexane, Rf = 0.39). The mixture was concentrated, resuspended in
35% EtOAc/65% hexane, and filtered to remove the bulk of the IBX
byproduct. The filtrate was concentrated, dissolved in CH2Cl2, and
washed with 10% aqueous Na2CO3. The organic layer was separated,
dried with anhydrous Na2SO4, filtered, and concentrated in vacuo.
Column chromatography (3% EtOH/97% CHCl3, Rf = 0.39) was
performed and provided the product 18 (0.75g, 79%). 1H NMR
(CDCl3): δ 10.21 (s, 3H, CHO), 8.64 (s, 3H, aromatic), which
matched the literature spectrum.20 Note: aldehyde 18 is also
commercially available from the Sigma-Aldrich Company.
ASSOCIATED CONTENT
* Supporting Information
■
S
1H spectra for known compound 1b, 1H and 13C NMR spectra
for compounds 6b, 7a−c, 8a, 8b, 11a−c, 15a, 15b, and 20b;
elemental analyses for 1b, 6a, 6b, 7a−c, 8a, 8b, 11, 15, and 20b,
and titration experiments with 6a in L3.6pl cells in the presence
of a fixed dose of DFMO and exogenous putrescine (or
spermine). This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (407) 823-6545. E-mail: Otto.Phanstiel@ucf.edu.
■
tert-Butyl (4-((4-Aminobutyl)(tert-butoxycarbonyl)amino)-
butyl)(methyl)carbamate 19. This compound was previously
reported by our group.17b
Notes
The authors declare no competing financial interest.
Tri-tert-butyl (((((Benzene-1,3,5-triyltris(methylene))tris-
(azanediyl))tris(butane-4,1-diyl))tris((tert-butoxycarbonyl)-
azanediyl))tris(butane-4,1-diyl))tricarbamate 20a. This com-
pound was synthesized previously.13b Briefly, Boc-protected homo-
spermidine 10a (0.72 g, 2 mmol) was dissolved with 25% MeOH/
CH2Cl2. 1,3,5-Triformylbenzene 18 (0.10 g, 0.63 mmol) was added to
the mixture and stirred for 3 h under a N2 atmosphere. Loss of the
ACKNOWLEDGMENTS
■
The authors thank the 2011 Department of Defense Congres-
sionally Directed Medical Research Program (CDMRP) Peer
Review Cancer Research Program (PRCRP) Discovery Award
CA110724 for financial support of this research and Dr. Isaiah
Fidler at the University of Texas M. D. Anderson Cancer
Center for the generous gift of L3.6pl cells for this research.
1
aldehyde CH signal at δ 10.21 was confirmed by H NMR (CDCl3).
The solvent was then removed under reduced pressure, and the crude
material was redissolved in 50% MeOH/CH2Cl2. To this new solution
was added NaBH4 (0.24 g, 6.3 mmol) at 0 °C, and the reaction was
stirred overnight at rt under a N2 atmosphere. Loss of the imine was
confirmed by 1H NMR. The solvent was then removed under reduced
pressure. A base workup was performed by redissolving the residue in
CH2Cl2 and washing with aqueous Na2CO3. The organic layer was
separated, dried over anhydrous Na2SO4, filtered, and concentrated to
give a yellow oil (1.13 g). Column chromatography (30% MeOH/69%
acetonitrile/1% NH4OH) was used to reisolate the unreacted amine
starting material (Rf = 0.7) and provide the trisubstituted aryl
compound 20a (0.35 g, 47%).13b 1H NMR (CDCl3): δ 7.14 (s, 3H,
aromatic), 4.69 (s, 3H, NH), 3.75 (s, 6H, CH2), 3.15 (br s, 18H,
ABBREVIATIONS USED
■
PTI, polyamine transport inhibitor; DFMO, α-difluoromethy-
lornithine; PTS, polyamine transport system; DFMO+PTI, α-
difluoromethylornithine used in combination with a polyamine
transport inhibitor; DFMO+Spd, α-difluoromethylornithine
used in combination with spermidine; DFMO+Spd+PTI, α-
difluoromethylornithine used in combination with spermidine
and a polyamine transport inhibitor; ODC, ornithine
decarboxylase; L-Lys-Spm, L-lysine spermine conjugate (used
361
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Journal of Medicinal Chemistry
Article
inhibitor-induced G1 cell cycle arrest in MYCN-amplified human
neuroblastoma cells. Oncogene 2005, 24, 5606−5618.
in this study); D-Lys-Spm = MQT 1426, D-lysine spermine
conjugate (not used in the study); SCC, squamous cell
carcinoma; PSV, polyamine sequestering vesicles; CHO,
Chinese hamster ovary; CHO-MG, Chinese hamster ovary
cells which are defective in polyamine transport; CHO-MG*,
Chinese hamster ovary cells which are defective in polyamine
transport and were derived from CHO-MG; AG, amino-
guanidine; Spd, spermidine; SOX, spermine oxidase; PAO,
polyamine oxidase; SSAT, spermidine-spermine acetyl trans-
ferase; IC10 value, the highest concentration of compound
which resulted in ≤10% cell growth inhibition (i.e., ≥90% cell
viability) vs an untreated control over the same time period;
IC50 value, the concentration of compound which resulted in
50% cell growth inhibition vs an untreated control over the
same time period; HBSS, Hank’s balanced salt solution; SDS,
sodium dodecyl sulfate
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