1-(N-alkylamino)-11-(N-ethylamino)-4,8-diazaundecanes: Simple synthetic polyamine analogues that differentially alter tubulin polymerization

Article abstract of DOI:10.1021/jm980603+

Polyamine analogues such as bis(ethyl)norspermine and N¹- (cyclopropylmethyl)-N¹¹-ethyl-4,8-diazaundecane (CPENSpm) act as potent modulators of cellular polyamine metabolism in vitro and possess impressive antitumor activity against

Full text of DOI:10.1021/jm980603+

J . Med. Chem. 1999, 42, 1415-1421
1415
1-(N-Alk yla m in o)-11-(N-eth yla m in o)-4,8-d ia za u n d eca n es: Sim p le Syn th etic
P olya m in e An a logu es Th a t Differ en tia lly Alter Tu bu lin P olym er iza tion
Heather K. Webb,∇ Zhiqian Wu,† Nilantha Sirisoma,† Hyo Chol Ha,§ Robert A. Casero, J r.,§ and
Patrick M. Woster*,†
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, Wayne State University,
Detroit, Michigan 48202, J ohns Hopkins Oncology Center, J ohns Hopkins University, Baltimore, Maryland 21231, and
Oridigm Corporation, Seattle, Washington 98109
Received October 28, 1998
Polyamine analogues such as bis(ethyl)norspermine and N1-(cyclopropylmethyl)-N11-ethyl-4,8-
diazaundecane (CPENSpm) act as potent modulators of cellular polyamine metabolism in vitro
and possess impressive antitumor activity against a number of cell lines. Some of these
polyamine analogues appear to produce their cell-type-specific cytotoxic activity through the
superinduction of spermidine/spermine N1-acetyltransferase (SSAT). However, there are several
analogues (e.g., N1-(cycloheptylmethyl)-N11-ethyl-4,8-diazaundecane (CHENSpm)) which are
effective cytotoxic agents but do not superinduce SSAT. We have previously demonstrated that
CPENSpm and CHENSpm both initiate the cell death program, although by different
mechanisms, and that CHENSpm (but not CPENSpm) induces a G2/M cell cycle arrest. We
now report that one potential mechanism by which some polyamine analogues can retard growth
and ultimately produce cytotoxicity is through interference with normal tubulin polymerization.
In these studies, we compare the effects of the polyamine analogues CHENSpm, CPENSpm,
and (S)-N1-(2-methyl-1-butyl)-N11-ethyl-4,8-diazaundecane (IPENSpm) on in vitro tubulin
polymerization. These spermine analogues behave very differently from spermine and from
each other in terms of tubulin polymerization rate, equilibrium levels, and time of polymeri-
zation initiation. These results demonstrate that structurally similar polyamine analogues with
potent antitumor effects can produce significantly different cellular effects. The discovery of
polyamine analogues that can alter tubulin polymerization provides a series of promising lead
compounds that may have a similar spectrum of activity to more difficult to synthesize
compounds typified by paclitaxel.
In tr od u ction
The natural polyamines serve a number of critical
functions in mammalian cells and are absolutely re-
quired for cellular division and viability.1 A number of
research groups, including our own,2-6 have described
the synthesis and evaluation of a series of bis(alkyl)-
substituted polyamine analogues with impressive in
vitro antitumor effects. These analogues have been
shown to enter the cell by the polyamine transport
system7 and then to disrupt the biosynthesis and
metabolic interconversion of cellular polyamines. In
various cultured tumor cell types, many of these agents
down regulate ODC and AdoMet-DC and superinduce
spermidine/spermine N1-acetyltransferase (SSAT), the
rate-limiting step in polyamine catabolism. However,
these bis-alkylated analogues do not substitute for the
F igu r e 1. Structures of the alkylpolyamine analogues
natural polyamines in terms of supporting cell growth.
The resultant cell is functionally depleted of polyamines,
a situation which results in rapid cytotoxicity in a
variety of tumor cell lines. One of the first of these
agents to be described, bis(ethyl)norspermine (BENSpm,
1; Figure 1), exhibits cell-type-specific cytotoxicity in
vitro against the human lung cancer cell lines NCI H157
(non-small-cell lung carcinoma) and NCI H82 (small-
cell lung carcinoma).8-11
BENSpm, CPENSpm, CHENSpm, and IPENSpm.
We previously described the synthesis and evaluation
of a series of unsymmetrically substituted alkylpolyamine
analogues, including CPENSpm (2) and CHENSpm (3),
shown in Figure 1, which exhibit significant antitumor
effects in vitro.2,3 Based on data gathered for these
analogues, our initial hypothesis was that the dif-
ferential induction of SSAT in the H157 line may play
a role in determining cell-type-specific sensitivity to the
bis-alkylated polyamine analogues. Although CHENSpm
treatment is cytotoxic in many of the same cell systems
as BENSpm and CPENSpm, CHENSpm cytotoxicity
* To whom correspondence should be addressed.
† Wayne State University.
§ J ohns Hopkins University.
∇ Oridigm Corp.
10.1021/jm980603+ CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/26/1999

1416 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8
Webb et al.
Sch em e 1
does not correlate with SSAT superinduction. These
data led us to search for the mechanisms of action
responsible for the cell-type-specific cytotoxicity caused
by 2 and the less cell-type-specific toxicity produced by
3 and to determine whether there are common inter-
mediates or endpoints produced by these agents. The
most significant common endpoint produced by 2 and 3
is the ability to induce programmed cell death (PCD).
Compound 2 induces PCD in both hormone-dependent
and hormone-independent breast tumor lines in vitro,12
as well as in the H157 non-small-cell lung tumor line.13
One component of this observed PCD appears to be
associated with the production of H2O2, secondary to
superinduction of SSAT by 2 and polyamine oxidase-
dependent oxidation of the resulting acetylated polyam-
ines.14 PCD induced by 2 is delayed but not abolished
in cells overexpressing the Bcl-2 antiapoptotic protein.
Although 3 does not cause superinduction of SSAT and
resultant overproduction of H2O2, it also induces PCD
in H157 lung tumor cells in vitro. As was the case for
analogue 2, PCD induced by 3 is delayed but not
abolished in cells overexpressing Bcl-2 and appears to
proceed by both caspase-3-dependent and -independent
mechanisms.15
Although both 2 and 3 induce PCD, it is likely that
they do so through a combination of potentially dis-
similar mechanisms. Consistent with this hypothesis is
the observation that they have dramatically different
effects on the cell cycle.14,15 Following 24-h treatment
of H157 non-small-lung carcinoma cells with 10 µM 2,
no significant effects on cell cycle are observed by flow
cytometric analysis. However, under the same condi-
tions, 10 µM 3 produces a dramatic G2/M cell cycle block
in normal and Bcl-2-overexpressing H157 cells.15 On the
basis of these observations, the potential mechanisms
leading to the ability of 3 to produce a G2/M block were
investigated. We now report the synthesis and evalua-
tion of the terminally alkylated polyamines CHENSpm
(3) and (S)-1-[N-(2-methyl-1-butyl)mino]-11-(N-ethyl-
amino)-4,8-diazaundecane (IPENSpm, 4). Each of these
analogues possesses the ability to significantly and
differentially alter tubulin polymerization in human
lung cancer cells.
Ch em istr y
The synthetic route leading to the target analogues
3 and 4 is outlined in Scheme 1. The tetramesitylated
N1-ethylnorspermine 5, prepared as previously de-
scribed,2 was treated with sodium hydride (60% oil
dispersion) in DMF, followed by addition of the ap-
propriate alkyl halide (cycloheptylmethyl iodide (6) or
(S)-(+)-1-bromo-2-methylbutane (7)), to afford the fully
protected precursor molecules 8 and 9, respectively.
Removal of the 2-mesitylenesulfonyl protecting group2,14,17
then afforded the crude target molecules, which were
purified by recrystallization from water/ethanol as the
tetrabromide salts 3 and (S)-4, respectively.
Effects on Cell Gr ow th a n d Cell Cycle
Each of the three analogues demonstrated similar
cytotoxic effects in the human non-small-cell lung
carcinoma line, NCI H157. In 96-h dose-response
studies each compound was found to be cytotoxic at
concentrations greater than 0.1 µM (Figure 2). However,
it should be noted that significant induction of SSAT
activity was only observed in cells treated with CPENS-
pm and not in cells treated with CHENSpm or IP-
ENSpm (Figure 3).
We have previously reported a differential effect on
the cell cycle by CHENSpm and CPENSpm.14,15 To
confirm these results and to compare the effects of the
newly synthesized IPENSpm, the effects of all three
compounds on the cell cycle progress were analyzed

1-(N-Alkylamino)-11-(N-ethylamino)-4,8-diazaundecanes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8 1417
F igu r e 2. Dose response to unsymmetrically substituted
polyamine analogues 2-4. NCI H157 cells were exposed to
increasing concentrations of the indicated compounds for 96
h. The growth effects are expressed as a ratio of cells at 96 h
(N1) versus the number of cells seeded at time zero (N). Cell
toxicity is defined as a N1/N ratio < 1. This figure is
representative of three separate experiments run in duplicate
with variation of 10% or less.
F igu r e 4. Effects of polyamine analogue treatment on the
cell cycle in NCI H157 cells. Subconfluent H157 cells were
exposed to A, no treatment; B, 10 µM CHENSpm; C, 10 µM
IPENSpm; D, 10 µM CPENSpm for 24 h. Cell cycle histograms
were derived as previously described.13 This figure depicts the
results of a minimum of three trials, each with nearly identical
results.
F igu r e 3. SSAT induction by polyamine analogues 2-4. NCI
H157 cells were treated with 10 µM of the indicated com-
pounds for 24 h followed by analysis of their SSAT activity.
Note the ordinate is a log scale. Only CPENSpm treatment
was found to superinduce SSAT. The graph depicts the results
of a representative experiment performed in triplicate, within
which the standard deviation was uniformly less than 10%.
F igu r e 5. Effects of CPENSpm (2), CHENSpm (3), IPENSpm
(4), and spermine on tubulin polymerization. Buffer containing
the polyamine was equilibrated at 37 °C. The polymerization
was initiated by the 10-fold dilution of 10 mg/mL tubulin which
had been thawed immediately before the reaction. The first
OD340 reading was taken at 30 s. Each point represents the
mean of 2-3 polymerization reactions; the values generally
varied <10%.
after a 24-h exposure to 10 µM of each compound
(Figure 4). As previously observed, CPENSpm treat-
ment had no significant effect on the cell cycle. However,
both CHENSpm and IPENSpm had a profound effect,
each treatment resulting in a significant G2/M block and
a concurrent decrease in the G1 fraction (Figure 4).
no detectable polymerization, as determined either by
increase in absorbance or by observable microtubule
formation (37 °C, MgCl2, without GTP or organic
solvent). In experiments run in the presence of spermine
or analogues 2-4, the three phases of tubulin poly-
merization (initiation, polymerization, and equilibrium)
were different. Initiation of polymerization was observ-
able following treatment with spermine and 4. Both
initiated polymerization approximately 1 min after
mixing tubulin with the buffer and the analogue. The
rate of polymerization was greatest in the case of 4 and
Effects on Tu bu lin P olym er iza tion
Analogues 2-4, as well as the natural tetraamine
spermine, stimulate tubulin polymerization in the ab-
sence of microtubule-associated proteins (MAPs) and
other polymerization stimulants (Figure 5 and Table 1).
In the absence of the analogues or spermine, there was

1418 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8
Webb et al.
Ta ble 1. Effects of Spermine, CPENSpm, CHENSpm, and IPENSpm on Tubulin Polymerization in the Presence and Absence of
MAPsa
rate
(∆OD340/min)
with MAPS
equilibrium OD340
(20-min reading)
with MAPS
rate
(∆OD340/min)
equilibrium OD340
(10-min reading)
compound
SPM (1 mM)
0.14
1.310
0.563
1.791
2.422
1.083
1.088
1.714
0.055
0.036
0.021
0.25
0.031
0.069
0.16
0.624
0.098
0.247
1.302
0.514
0.392
1.007
CPENSpm (1 mM)
0.096
0.69
IPENSpm (1 mM)
CHENSpm (1 mM)
CPENSpm (1 mM) + SPM (1 mM)
IPENSpm (1 mM) + SPM (1 mM)
CHENSpm (1 mM) + SPM (1 mM)
0.37
0.131
0.205
0.131
a
Each reported data point is the result of at least three determinations which in all cases varied by 10% or less.
F igu r e 6. Effects of CPENSpm (2), CHENSpm (3), IPENSpm
(4), and spermine on the polymerization of MAP-rich tubulin.
The buffer containing polyamine was equilibrated at 37 °C.
The polymerization was initiated by 7.5-fold dilution of 7.5 mg/
mL MAP-rich tubulin which had been thawed just prior to the
reaction. The first OD340 reading was taken at 30 s. Each point
represents the mean of 2-3 polymerization reactions; the
values generally varied <10%.
F igu r e 7. Immunohistochemistry of NCI H157 cells probed
using anti-tubulin monoclonal antibody. The nucleus was
stained with methyl green. These are representative pictures
of three separate experiments. The DAB staining was per-
formed simultaneously to ensure comparable results: left,
control; right, 24-h treatment with 10 µM CHENSpm (3).
proceeded 4.9 times faster than that of spermine. The
analogues altered the equilibrium OD340 from the
control in the following order: 3 > 4 > spermine > 2.
The samples with OD340 values of 0.7 or greater showed
that an increasing portion of the material causing
turbidity was nonmicrotubule aggregates and bundled
microtubules. These were identified when the poly-
merization was performed with rhodamine-modified
tubulin and observed by fluorescent microscopy.
To determine if the presence of spermine would
mitigate the effects of analogues, the effect of spermine
on the polymerization reaction was observed (Table 1).
Polymerization rates are significantly enhanced when
3 or 4 is added to the polymerization reaction, while 2
has an effect similar to that of spermine. An analogous
effect is seen on the equilibrium value, since 3 and 4
enhance tubulin polymerization, while 2 diminishes the
effect with respect to control. When analogues 2-4 are
added to the mixture in combination with 1 mM
spermine, the rate and equilibrium values for tubulin
polymerization did not differ significantly from control
values. This strongly suggests that analogues 2-4 are
competing for spermine at some polymerization-induc-
ing site on tubulin. Since the concentration of polyca-
tions is double in each of the combination reactions, the
increase in polymerization is likely a specific effect
rather than the result of simply increasing polycations
in the reaction.
different in the presence of MAPs. Under the conditions
of this assay, again with MgCl2 and without GTP or
organic solvent, there is observable microtubule forma-
tion. The polymerization profile changed most signifi-
cantly for 4 relative to the reaction without MAPs.
Compound 3 and spermine showed decreases in their
ability to effect the tubulin polymerization. The initia-
tion of polymerization was delayed substantially, and
the rate of polymerization was the most significantly
decreased by the presence of MAPs. Compound 3 was
most effective at altering the tubulin dynamics in the
presence of both MAPs and spermine and increased both
the rate and the equilibrium relative to the reaction
with spermine and MAPs only.
Compound 3 altered the microtubule density in the
putative centrosome adjacent to the nucleus when
analyzed by immunohistochemical staining of tubulin
(Figure 7). The cytoplasmic microtubules do not appear
to be affected by treatment. However, the strong stain-
ing of the microtubules emanating from the centrosomal
area is absent or significantly reduced. Higher concen-
trations of 3 caused rapid apoptosis and thus were not
amenable to immunohistochemistry. Compound 4 was
also tested for its ability to alter the cellular microtu-
bules. There were no observable changes in the density
or distribution of the microtubules after treatment with
compound 4.
To further investigate elements that could compete
for interaction with tubulin in the intact cell, the
polymerization assay was repeated with MAP-rich tu-
bulin (Figure 6 and Table 1). MAPs are believed to
interact with the tubulin at least in part through their
polylysine cationic regions. A direct comparison of the
OD340 values is not valid since the concentration of
tubulin and the diameter of the microtubules are
Discu ssion
Interference with polyamine metabolism has been
established as a rational target for antineoplastic
intervention. Much work has been performed aimed at

1-(N-Alkylamino)-11-(N-ethylamino)-4,8-diazaundecanes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8 1419
the inhibition of polyamine biosynthesis and, more
recently, to induce polyamine catabolism. The net effect
of these strategies has been to suggest that growth
inhibition is concurrent with the depletion of natural
polyamines. However, the underlying mechanisms in-
volved in the inhibition of growth by the disturbance of
polyamine metabolism are only now being elucidated.
We have demonstrated that in some cases, the super-
induction of SSAT and the subsequent production of
H2O2 lead to the induction of programmed cell death.14,15
However, it is clear that polyamine analogues can kill
cells through other mechanisms. In the current work,
we present evidence that two new cytotoxic antitumor
polyamine analogues that do not superinduce SSAT
produce a significant G2/M block and effect the process
of tubulin polymerization. These results underscore the
observation that small changes in analogue structure
can have profound effects on their activity. It should be
noted that Kramer et al. have reported a G1 block
produced by several symmetrically substituted polyamine
analogues.18 However, the underlying mechanisms caus-
ing the observed G1 block are currently not known.
CHENSpm and IPENSpm could be affecting tubulin
polymerization by different mechanisms.
In the in vitro tubulin polymerization assay, micro-
tubules do not spontaneously form without the addition
of a stimulant such as paclitaxel or, in this case, a
polyamine or MAPs. However, the final optical density
at the equilibrium stage with 1 mM polyamine analogue
added is higher than would be expected for normal
helical microtubules. Other forms of polymerized tubulin
have been found to form in the presence of cations, such
as “C” tubules, crenelated ribbon, and “σ” forms.19 These
forms of tubulin defract light more effectively than
regular microtubules. We are unable to distinguish
these forms using fluorescence microscopy of rhodamine-
modified tubulin. However, we can observe linear forms
of tubulin polymer and the associated increase in the
amount of these linear tubulin forms observed in
bundles with time and polyamine concentration. The
observed increase in bundling with time occurs whether
the polymerization reaction takes place at 25 or 4 °C.
The observable effects of polyamine analogues on
tubulin dynamics in a cuvette are more dramatic than
the effects observed in the cell, although this is probably
due to the higher (1 mM) concentrations used for these
assay procedures. However, significant effects on tubu-
lin polymerization can be detected at levels of the
analogues that are closer to cytotoxic concentrations.
Pohjanpelto et al. observed a large decrease in the total
microtubule content of polyamine-deficient CHO cells
using immunohistochemistry.21 Following treatment
with 10 µM analogue concentrations, immunohistochem-
ical studies indicate that these polyamine analogues do
not appear to decrease the quantity of microtubules but
rather decrease the association of microtubules with the
centrosomes. This is consistent with G2/M arrest, since
abnormal association of microtubules with the cen-
trosomes could prevent spindle formation and mitosis.
IPENSpm did not appear to alter cellular microtubule
structure despite the observed G2/M arrest, again sug-
gesting that IPENSpm and CHENSpm have differential
mechanisms for inducing tubulin polymerization changes.
It is also possible that alterations in the microtubules
were not observable due to the competing apoptotic
mechanisms induced by IPENSpm.
Both R- and â-tubulin have an acidic carboxy termi-
nus which has been proposed to inhibit polymeriza-
tion.19 Natural cations serve to affect the microtubule
polymerization dynamics, both enhancing and inhibiting
microtubule formation. The effects of Ca2+ and Mg2+ on
microtubule dynamics are well-documented; however,
there are other natural cations that may play a role in
microtubule dynamics. Deprivation of polyamines has
been shown to have significant effects on the cytoskel-
eton in some cells.20-22 In 1985, it was observed that
natural polyamines enhance tubulin polymerization in
the presence and absence of MAPs.22 Recently, Wolff
proposed that spermidine and spermine may affect the
state of tubulin polymerization and promote microtubule
assembly.23 Using a modification of the polymerization
assay described here, in which DMSO or taxol and 0.8
mM GTP are used to promote polymerization, Wolff
observed that 100 µM spermine tripled the rate of
polymerization. The stabilization of microtubule forma-
tion by the natural polyamines, spermine and spermi-
dine, may be part of the natural dynamics of microtu-
bules and represents part of the influence of polyamines
on cell cycle.
The results of the current study demonstrate that
small changes in analogue structure can have profound
consequences on observed cellular effects. Compound
2-induced toxicity appears to be, at least in part,
associated with its induction of SSAT14 without obvious
effects on cell cycle progression. By contrast, neither 3
nor 4 superinduce SSAT but both produce a profound
G2/M block and subsequent cytotoxicity by an apparent
PCD pathway.15 Although the observed changes in
tubulin polymerization induced by 3 and 4 suggest a
direct effect on the polymerization process, the possibil-
ity that these effects are caused by a combination of
other mechanisms, including an analogue-induced al-
teration in specific gene expression, cannot be ex-
cluded.15 Further study will be necessary to determine
what properties of terminally alkylated polyamine
analogues should be synthetically exploited to improve
tumor-specific activity. The synthesis and evaluation of
additional analogues, as well as additional experiments
The series of polyamine analogues presented here
indicate that there is a more specific interaction of
polyamines with tubulin than a simple electrostatic
interaction, since there are significant changes in the
initiation of polymerization, polymerization rate, and
equilibrium OD340 when different alkyl groups are
appended to the analogues. The three analogues
CHENSpm, IPENSpm, and CPENSpm have identical
point charge distributions along their alkyl backbone.
The only structural differences between these analogues
is the alkyl modification at the one terminal nitrogen.
Though IPENSpm and CHENSpm both induce a G2/M
arrest, only CHENSpm shows an alteration in the
immunohistochemical results. This is consistent with
the observation that IPENSpm is not inducing an
increase in polymerization rate and equilibrium values
in the in vitro polymerization assay in the presence of
MAPs. IPENSpm also elicits a less pronounced effect
on cell cycle. Taken together, these results suggest that

1420 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8
Webb et al.
(S)-1-[N-(2-Met h yl-1-b u t yl)-N-(2-m esit ylen esu lfon yl)-
am in o]-4,8-N-(2-m esitylen esu lfon yl)-11-[N-eth yl-N-(2-m es-
itylen esu lfon yl)a m in o]-4,8-d ia za u n d eca n e (9). A 0.053-g
portion of sodium hydride (60% mineral oil dispersion, 0.0022
mol) in 10 mL of dry DMF was added slowly via dry syringe
to a solution of 5 (1.0 g, 0.0011 mol) and (S)-2-methylbromobu-
tane (7) (0.498 g, 0.0033 mol) in 20 mL of dry DMF at 0 °C
under a nitrogen atmosphere. The reaction was conducted
exactly as described above for the synthesis of compound 8 to
afford pure 9 (0.815 g, 73%) as a yellow oil: 1H NMR (CDCl3
) δ 6.90 (s, 8H, mesityl aromatic H3 and H5), 3.25-3.03 (br m,
17H, H1, H3, H5, H7, H9, H11, 2-methylbutyl methyne, 2-me-
thylbutyl N-CH2 and ethyl CH2), 2.31 (s, 24H, mesityl 2-CH3
and mesityl 6-CH3), 2.27 (s, 12H, mesityl 4-CH3), 1.67 (m, 8H,
H2, H6, H10 and 2-methylbutyl CH2), 1.21 (s, 6H, 2-methylbutyl
CH3), 1.03 (t, 3H, ethyl CH3). Anal. (C52H78N4O8S4) C, H, N.
1-[N-(Cycloh ep t ylm et h yl)a m in o]-11-(N-et h yla m in o)-
4,8-d ia za u n d eca n e Tetr a h yd r obr om id e, 3. An 11.01-g
portion of phenol (0.117 mol) was dissolved in 50 mL of 30%
HBr/HOAc in a stoppered flask, and to this mixture was added
a solution of 8 (0.978 g, 0.00093 mol) in 20 mL of ethyl acetate
in three portions over a period of 3 h. After the addition was
complete, the reaction mixture was stirred for an additional
15 h at room temperature, then cooled to 0 °C, and diluted
with 100 mL of water. The aqueous phase was washed with
two 100-mL portions of ethyl acetate before being lyophilized
to give the crude product as dark yellow solid. This crude
product was washed with methanol and filtered to yield the
tetrahydrobromide salt of 3 (0.349 g, 57.8%) as a white solid.
An analytical sample of 3 was prepared by recrystallization
from aqueous ethanol: 1H NMR (D2O) δ 3.14 (m, 14H, H1, H3,
H5, H7, H9, H11 and ethyl CH2), 2.93 (d, 2H, cycloheptylmethyl
methylene), 2.09 (m, 8H, H2, H6, H10 and cycloheptylmethyl
methylene), 1.41-1.76 (br m, 12H, remaining cycloheptyl
protons), 1.25 (t, 2H, ethyl CH3). Anal. (C19H46N4Br4) C, H, N.
(S)-1-[N-(2-Meth yl-1-bu tyl)a m in o]-11-(N-eth yla m in o)-
4,8-d ia za u n d eca n e Tetr a h yd r obr om id e, 4. A 9.51-g por-
tion of phenol (0.101 mol) was dissolved in 50 mL of 30% HBr/
HOAc in a stoppered flask, and to this mixture was added a
solution of 9 (0.815 g, 0.0008 mol) in 20 mL of ethyl acetate in
three portions over a period of 3 h. The reaction was conducted
exactly as described for the synthesis of 3 to yield the
tetrahydrobromide salt of 4 (0.462 g, 85%) as a white solid.
An analytical sample of 4 was prepared by recrystallization
from aqueous ethanol: 1H NMR (D2O) δ 3.08 (m, 15H, H1, H3,
H5, H7, H9, H11, ethyl CH2 and 2-methylbutyl methyne), 2.95
and 2.82 (pair of m, 2H, diastereotopic2-methylbutyl N-CH2),
2.04 (m, 6H, H2, H6, and H10) 1.72 and 1.36 (pair of m, 2H,
diastereotopic 2-methylbutyl C-CH2), 1.21 (t, 3H, 2-methyl-
butyl C-CH3), 0.90 (d, 3H, 2-methylbutyl CH3), 0.83 (t, 3H,
ethyl CH3). Anal. (C16H42N4Br4) C, H, N.
Cell Cu ltu r e Stu d ies. The NCI H157 non-small-cell lung
carcinoma line was maintained in culture as previously
described.24 This line was refed with fresh medium every 3
days to maintain log phase growth. Cells were exposed to the
polyamine analogue N1,N12-bis(ethyl)spermine and the poten-
tial SSAT inhibitor. Effects on growth and SSAT activity were
then determined as previously published.3 The SSAT activity
of these cell lysates was determined by a previously published
method.10
aimed at elucidation of the cellular effects of these
analogues, are ongoing concerns in our laboratories.
Exp er im en ta l Section
All reagents were purchased from Aldrich Chemical Co.
(Milwaukee, WI) or Sigma Chemical Co. and were used
without further purification except as noted below. Triethyl-
amine was distilled from potassium hydroxide and stored in
a nitrogen atmosphere. Methanol was distilled from magne-
sium and iodine under a nitrogen atmosphere and stored over
molecular sieves. Methylene chloride was distilled fron phos-
phorus pentoxide, and chloroform was distilled from calcium
sulfate. Dimethyl formamide was dried by distillation from
anhydrous calcium sulfate and was stored under nitrogen.
Preparative scale chromatographic procedures were carried out
using E. Merck silica gel 60, 230-440 mesh. Thin-layer
chromatography was conducted on Merck precoated silica gel
60 F-254. All 1H and 13C NMR spectra were recorded on a
General Electric QE-300 spectrometer, and all chemical shifts
are reported as δ values referenced to TMS or DSS. Infrared
spectra were recorded on a Nicolet 5DXB FT-IR spectropho-
tometer and are referenced to polystyrene. In all cases, 1H
NMR, 13C NMR, and IR spectra were consistent with assigned
structures. Melting points were recorded on a Thomas-Hoover
capillary melting point apparatus and are uncorrected. Mi-
croanalyses were performed by Galbraith Laboratories, Knox-
ville, TN, and were within 0.4% of calculated values.
Cycloh ep tylm eth yl Iod id e, 6. A 5.0-g portion of cyclo-
heptanemethanol (0.039 mol) was dissolved in 100 mL of dry
CH2Cl2 along with 4.76 g (0.047 mol) of triethylamine and 5.38
g (0.047 mol) of methanesulfonyl chloride, and the mixture was
allowed to stir at room temperature overnight. The reaction
mixture was washed with two 100-mL portions of 1 N HCl,
100 mL of water, and 100 mL of saturated NaCl, and the
organic layer was then dried over MgSO4. Filtration and
removal of solvent afforded the crude mesylate (7.88 g, 97.9%)
as a colorless oil. The crude product was sufficiently pure to
use in the subsequent reaction without further purification.
A 7.88-g portion of the crude mesylate (0.038 mol) was
dissolved in 100 mL of acetone, and 86.20 g (0.575 mol) of
sodium iodide was added. The reaction mixture was allowed
to reflux for 3 h, after which the solution was evaporated to
dryness and redissolved in 100 mL of ethyl acetate. The
organic layer was washed with 100 mL of water and 100 mL
of saturated NaHCO3 and then dried over MgSO4. Filtration
and removal of the solvent in vacuo afforded the crude iodide
6, which was chromatographed on silica gel (hexanes) to
produce 7.05 g (77.5%) of the desired iodide as a pale pink oil:
1H NMR (CDCl3) δ 3.13 (d, 2H, exocyclic CH2), 1.78 (m, 2H,
H2 and H7 cis to I), 1.62 (m, 3H, H1 methyne, H2 and H7 trans
to I), 1.49 (m, 6H, H3, H6 and H4 and H5 cis to I), 1.26 (m, 2H,
H4 and H5 trans to I); 13C NMR (CDCl3) δ 41.39 (C1), 34.67 (C2
and C7), 28.13 (C4 and C5), 26.08 (C3 and C6), 18.04 (exocyclic
CH2).
1-[N-(Cycloh ep t ylm et h yl)-N-(2-m esit ylen esu lfon yl)-
am in o]-4,8-N-(2-m esitylen esu lfon yl)-11-[N-eth yl-N-(2-m es-
itylen esu lfon yl)a m in o]-4,8-d ia za u n d eca n e (8). A 0.053-g
portion of sodium hydride (60% mineral oil dispersion, 0.0022
mol) in 10 mL of dry DMF was added slowly via dry syringe
to a solution of 5 (1.0 g, 0.0011 mol) and cycloheptylmethyl
iodide (6) (0.786 g, 0.0033 mol) in 20 mL of dry DMF at 0 °C
under a nitrogen atmosphere. The reaction was allowed to stir
at 0 °C for 30 min, and then cold water (10 mL) and chloroform
(30 mL) were added with stirring. Removal of the solvent in
vacuo (35 °C at 0.2 mmHg) and chromatography of the residue
on silica gel (hexane/ethyl acetate, 5:5) then afforded pure 8
(0.978 g, 84%) as a yellow oil: 1H NMR (CDCl3) δ 6.93 (s, 8H,
mesityl aromatic H-3 and H5), 3.18 (q, 2H, ethyl CH2), 3.00
(m, 13H, H1, H3, H5, H7, H9, H11 and cycloheptylmethyl H1
methyne), 2.77 (m, 2H, cycloheptylmethyl exocyclic methyl-
ene), 2.56 (m, 24H, mesityl 2-CH3 and 6-CH3), 2.29 (s, 12H,
mesityl 4-CH3), 1.63 (m, 6H, H2, H6 and H10), 1.45 (m, 4H,
cycloheptylmethyl H2 and H7), 1.19 (m, 8H, cycloheptylmethyl
H3, H4, H5 and H6), 0.9 (t, 3H, ethyl CH3). Anal. (C55H82N4O8S4)
C, H, N.
Tu bu lin P olym er iza tion Assa y. Tubulin polymerization
was monitored by the change in optical density at 340 nm
using a modification of the method described by J ordan et al.19
Purified bovine brain tubulin with or without MAPs or glycerol
was purchased from Cytoskeleton (Denver, CO). The final
buffer concentrations for tubulin polymerization were 0.1 M
2-(N-morpholino)ethanesulfonic acid (MES) (pH 6.7), 1.0 mM
MgCl2, 0.5 mM ethylene glycol bis(â-aminoethyl ether)
N,N,N′,N′-tetraacetic acid (EGTA) (buffer A). Test analogues
were added in 1 mM concentration, and then all components
except the purified tubulin were warmed to 37 °C. The reaction
was initiated by the addition of tubulin to a final concentration
of 1.0 mg/mL (10 µM heterodimer for pure tubulin and
approximately 7 µM for the MAP-rich tubulin). The optical

1-(N-Alkylamino)-11-(N-ethylamino)-4,8-diazaundecanes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8 1421
(8) Casero, R. A.; Ervin, S. J .; Celano, P.; Baylin, S. B.; Bergeron,
R. J . Differential response to treatment with bis(ethyl)polyamine
analogues between human small cell lung carcinoma and undif-
ferentiated large cell lung carcinoma in culture. Cancer Res.
1989, 49, 639-643.
(9) Casero, R. A.; Celano, P.; Ervin, S. J .; Porter, C. W.; Bergeron,
R. J .; Libby, P. R. Differential induction of spermidine/spermine-
N1-acetyltransferase in human lung cancer cells by the bis-
(ethyl)polyamine analogues. Cancer Res. 1989, 49, 3829-3833.
(10) Casero, R. A.; Mank, A. R.; Xiao, L.; Smith, J .; Bergeron, R. J .;
Celano, P. Steady-state messenger RNA and activity correlates
with sensitivity to N1,N12-bis(ethyl)spermine in human cell lines
representing the major forms of lung cancer. Cancer Res. 1992,
52, 5359-5363.
(11) Casero, R. A.; Celano, P.; Ervin, S. J .; Wiest, L.; Pegg, A. E. High
specific induction of spermidine/spermine-N1-acetyltransferase
in a human large cell lung carcinoma. Biochem. J . 1990, 270,
615-620.
(12) McClosky, D. E.; Casero, R. A., J r.; Woster, P. M.; Davidson, N.
E. Induction of programmed cell death in human breast cancer
cells by an unsymmetrically alkylated polyamine analogue.
Cancer Res. 1995, 55, 3233-3236.
(13) McClosky, D. E.; Yang, J .; Woster, P. M.; Davidson, N. E.;
Casero, R. A., J r. Polyamine analogue induction of programmed
cell death in human lung tumor cells. Clin. Cancer Res. 1996,
2, 441-446.
(14) Ha, H. C.; Woster, P. M.; Yager, J . D.; Casero, R. A., J r. The
role of polyamine catabolism in polyamine analogue-induced
programmed cell death. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
11557-11562.
(15) Ha, H. C.; Woster, P. M.; Casero, R. A., J r. Unsymmetrically
substituted polyamine analogue induces caspase-independent
programmed cell death in Bcl-2-overexpressing cells. Cancer Res.
1998, 58, 2711-2714.
(16) Yajima, H.; Takeyama, M.; Kanaki, J .; Nishimura, O.; Fujino,
M. Studies on Peptides. LXXX. NG-Mesitylene-2-sulfonylargin-
ine. Chem. Pharm. Bull. 1978, 26, 3752-3757.
density was measured by a Perkin-Elmer Lambda 4B UV/VIS
spectrophotometer with a temperature-controlled cuvette holder.
Micr otu bu le Micr oscop y. Purified bovine brain tubulin
without MAPs or glycerol and modified with rhodamine was
purchased from Cytoskeleton (Denver, CO). The rhodamine-
modified bovine brain tubulin was diluted to a stoichiometry
of 0.04 rhodamine modifications per heterodimer. A solution
was prepared with buffer A. The tubulin was added, and the
solution was incubated at room temperature for 1 h. The
resulting microtubules were observed using fluorescence mi-
croscopy.
Im m u n oh istoch em istr y. NCI H157 cells were grown on
a single-well glass slide in RPMI 1640 with 9% fetal calf serum,
100 units/mL penicillin, and 100 units of streptomycin. The
cells were then exposed to 1.0 mM aminoguanidine with and
without 10 µM 3 or 4 for 24 h. The cells were washed with
PBS, fixed with 70% ethanol at -20 °C, and permeablized with
0.1% Triton X-100 and 0.1% BSA in PBS. The slides were then
covered with the monoclonal TUB 2.1 anti-â-tubulin (Sigma,
St. Louis, MO). The slides were washed with PBS again and
covered with the secondary antibody, a rabbit anti-mouse HRP
conjugate antibody (Sigma). The tubulin antibody was then
visualized by Elite Universal kit Vectastain ABC peroxidase
staining method (Vector Laboratories, Inc., Burlingame, CA)
followed by Sigma FAST DAB (3,3′-diaminobenzidine tetrahy-
drochloride) staining. All slides were stained for 1 min to
ensure comparable results.
Cell Cycle An a lysis. NCI H157 cells (0.5 × 106) were
plated in a 25-mm2 flask and allowed to adhere. The cells were
then treated with 10 µM of each analogue in the presence of
1 mM aminoguanidine for 24 h before harvesting. Flow
cytometric analyses of cells treated for 24 h with the indicated
compounds were performed as previously described.25
(17) J asys, V. J .; Kelbaugh, P. R.; Nason, D. M.; Phillips, D.; Rosnack,
K. J .; Forman, J . T.; Saccomano, N. A.; Stroh, J . G.; Volkmann,
R. A. Novel Quaternary Ammonium Salt-Containing Polyamines
from the Agelenopsis aperta Funnel-Web Spider. J . Org. Chem.
1992, 57, 1814-1820.
(18) Kramer, D. L.; Fogel-Petrovic, M.; Diegelman, P.; Cooley, J . M.;
Bernacki, R. J .; McManis, J . S.; Bergeron, R. J .; Porter, C. W.
Effects of polyamine analogues on cell cycle progression and
apoptosis in MALME-3M human melanoma cells. Cancer Res.
1997, 57, 5521-5527.
Ack n ow led gm en t. This work was supported by
NIH Grants RO1 CA63552 (P.M.W.), CA58184 (R.A.C.),
and RO1 CA51085 (R.A.C.). The excellent technical
assistance of Ms. Michelle Boahbedason and Ms. Alexis
Mesa is gratefully acknowledged.
Refer en ces
(19) J ordan, M. A.; Toso, R. J .; Thrower, D.; Wilson, L. Mechanism
of mitotic block and inhibition of cell proliferation by taxol at
low concentrations. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9552-
9556.
(1) For more information on the function of the natural polyamines
and the consequences of inhibition of the polyamine pathway,
see: A Guide to the Polyamines; Cohen, S., Ed.; Oxford Uni-
versity Press: New York, 1998; Chapter 23, 624 pp. See also:
Casero, R. A.; Pegg, A. E. Spermidine/spermine-N1-acetyltrans-
ferase - the turning point in polyamine metabolism. FASEB J .
1993, 7, 653-661. Pegg, A. E. Polyamine metabolism and its
importance in neoplastic growth and as a target for chemo-
therapy. Cancer Res. 1988, 48, 759-774.
(2) Saab, N. H.; West, E. E.; Bieszk, N. C.; Preuss, C. V.; Mank, A.
R.; Casero, R. A.; Woster, P. M. Synthesis and Evaluation of
Unsymmetrically Substituted Polyamine Analogues as Inhibitors
of Spermidine/Spermine-N1-Acetyltransferase (SSAT) and as
Potential Antitumor Agents. J . Med. Chem. 1993, 36, 2998-
3004.
(3) Casero, R. A.; Mank, A. R.; Saab, N. H.; Wu, R. H.; Woster, P.
M. Growth and Biochemical Effects of Asymmetrically Substi-
tuted Polyamine Analogues in Human Lung Tumor Cells.
Cancer Chemother. Pharmacol. 1995, 36, 69-74.
(4) Bergeron, R. J .; Neims, A. H.; McManis, J . S.; Hawthorne, T.
R.; Vinson, J . R. T.; Bortell, R.; Ingeno, M. J . Synthetic
Polyamine Analogues as Antineoplastics. J . Med. Chem. 1988,
31, 1183-1190.
(5) Bergeron, R. J .; McManis, J . S.; Liu, C. Z.; Feng, Y.; Weimar,
W. R.; Luchetta, G. R.; Wu, Q.; Ortiz-Ocasio, J .; Vinson, J . R.
T.; Kramer, D.; Porter, C. Antiproliferative properties of polyamine
analogues: A structure-activity study. J . Med. Chem. 1994, 37,
3464-3476.
(6) Edwards, M. L.; Prakash, N. J .; Stemerick, D. M.; Sunkara, S.
P.; Bitonti, A. J .; Davis, G. F.; Dumont, J . A.; Bey, P. Polyamine
analogues with antitumor activity. J . Med. Chem. 1990, 33,
1369-1375.
(7) Porter, C. W.; Ganis, B.; Vinson, T.; Marton, L. J .; Kramer, D.
L.; Bergeron, R. J . A comparison and characterization of growth
inhibition by R-difluoromethylornithine (DFMO), an inhibitor
of ornithine decarboxylase, and N1,N8-bis(ethyl)spermidine (BES),
an apparent regulator of the enzyme. Cancer Res. 1986, 46,
6279-6285.
(20) De Brabander, M.; Geuens, G.; Nuydens, R.; Willebrords, R.; De
Mey, J . Taxol induces the assembly of free microtubules in living
cells and blocks the organizing capacity of the centrosomes and
kinetochores. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 5608-5612.
(21) (a) Phojanpelto, P.; Virtanen, I.; Holtta, E. Polyamine starvation
causes disappearance of actin filaments and microtubules in
polyamine-auxotrophic CHO cells. Nature 1981, 293, 475-477.
(b) Pomidor, M. M.; Ruhl, K. K.; Zheng, P.; Song, Y.; J anne, O.
A.; Tuan, R. S.; Hickok, N. J . Relationship between ornithine
decarboxylase and cytoskeletal organization in cultured human
keratinocytes: cellular responses to phorbol esters, cytochala-
sins, and R-difluoromethylornithine. Exp. Cell Res. 1995, 221,
426-437. (c) Sunkara, P. S.; Rao, P. N.; Nishioka, K.; Brinkley,
B. R. Role of polyamines in cytokinesis of mammalian cells. Exp.
Cell Res. 1979, 119, 63-68.
(22) Anderson, P. J .; Bardocz, S.; Campos, R.; Brown, D. L. The effect
of polyamines on tubulin assembly. Biochem. Biophys. Res.
Commun. 1985, 132, 147-154.
(23) Wolff, J . Promotion of microtubule assembly by oligocations:
cooperativity between charged groups. Biochemistry 1998, 37,
10722-10729. Wolff, J .; Knipling, L.; Sackett, D. L. Charge-
shielding and the “paradoxical” stimulation of tubulin poly-
merization by guanidine hydrochloride. Biochemistry 1996, 35,
5910-5920.
(24) Casero, R. A.; Go, B.; Theiss, H. W.; Smith, J .; Baylin, S. B.;
Luk, G. D. Cytotoxic response of the relatively difluoromethyl-
ornithine-resistant human lung tumor cell line NCI H157 to the
polyamine analogue N1,N8-bis(ethyl)spermidine. Cancer Res.
1987, 47, 3964-3967.
(25) Dietch, A. D.; Law, H.; deVere White, R. A stable propidium
iodide staining procedure for flow cytometry. J . Histochem.
Cytochem. 1982, 30, 967-972.
J M980603+