Conformationally restricted analogues of 1N,14N-bisethylhomospermine (BE-4-4-4): Synthesis and growth inhibitory effects on human prostate cancer cells

Article abstract of DOI:10.1021/jm000309t

Twelve analogues of ¹N, ¹⁴N-Bisethylhomospermine (BE-4-4-4) with restricted conformations were synthesized in the search for cancer chemotherapeutic agents with higher cytotoxic activities and lower systemic toxicities than BE-4-4-4.

Full text of DOI:10.1021/jm000309t

390
J . Med. Chem. 2001, 44, 390-403
1
Con for m a tion a lly Restr icted An a logu es of N,14N-Biseth ylh om osp er m in e
(BE-4-4-4): Syn th esis a n d Gr ow th In h ibitor y Effects on Hu m a n P r osta te Ca n cer
Cells
Aldonia Valasinas, Aparajita Sarkar, Venodhar K. Reddy, Laurence J . Marton, Hirak S. Basu,* and
Benjamin Frydman*
SLIL Biomedical Corp., 535 Science Drive, Suite C, Madison, Wisconsin 53711
Received J uly 21, 2000
1
Twelve analogues of N,14N-bisethylhomospermine (BE-4-4-4) with restricted conformations
were synthesized in the search for cancer chemotherapeutic agents with higher cytotoxic
activities and lower systemic toxicities than BE-4-4-4. The central butane segment of BE-4-
4-4 was replaced with a 1,2-substituted cyclopropane ring, a 1,2-substituted cyclobutane ring,
and a 2-butene residue. In each case, the cis/trans-isomeric pair was synthesized. Cis-
monounsaturation(s) was also introduced at the outer butane segment(s) of BE-4-4-4. The two
possible cis-dienes and a cis-triene formally derived from the tetraazaeicosane skeleton of BE-
4-4-4 were also prepared. Four cultured human prostate cancer cell lines (LnCap, DU145,
DuPro, and PC-3) were treated with the new tetramines to examine their effects on cell growth
with a MTT assay. One representative cell line (DuPro) was selected to further study the cellular
uptake of the novel tetramines, their effects on intracellular polyamine pools, and their
cytotoxicity. All tetramines entered the cells, reduced cellular putrescine and spermidine pools
while exerting only a small effect on the spermine pool, inhibited cell growth, and killed 2-3
logs of cells after 6 days of treatment at 10 µM. Four new tetramines, the two cyclopropyl
isomers, the trans-cyclobutyl isomer, and the (5Z)-tetraazaeicosene, were more cytotoxic than
their saturated counterpart (BE-4-4-4). Their cytotoxicity, however, could not be correlated
either with their cellular uptake or with their ability to deplete intracellular polyamine pools.
We attribute their cytotoxicity to their specific molecular structures. The cytotoxicity was
markedly reduced when the central butane segment was deprived of its rotational freedom by
replacing it with a double bond. Introduction of a triple bond or a benzene-1,2-dimethyl residue
at the central segment of the polyamine chain, led to complete loss of biological activity. The
conformationally restricted alicyclic derivatives were not only more cytotoxic than was the freely
rotating BE-4-4-4 by several orders of magnitude but also had much lower systemic toxicities
than the latter. Thus, we obtained new tetramines with a wider therapeutic window than BE-
4-4-4.
In tr od u ction
therapeutic activity,6 and cis-unsaturated derivatives
have been prepared to facilitate their metabolic break-
down by mixed-function oxidases.7
N-Alkylated analogues of the natural polyamines
(spermine, spermidine, and putrescine) exhibit strong
cytotoxic activity against human tumor cell lines.1 It is
The design of new and more efficacious tetramines
poses drawbacks while offering tempting advantages.
The main drawback in the search of tetramines with
wider therapeutic windows is that the precise mecha-
nisms by which they kill tumor cells and cause systemic
toxicity are still not entirely clear.8 It has been estab-
lished that polyamines and their analogues bind to
nucleic acids and alter their conformations,9 that they
bind to receptor targets,10 that they drastically reduce
the level of ornithine decarboxylase11,12 (the first enzyme
in the pathway that leads to spermine biosynthesis in
mammals), that they upregulate the levels of spermi-
dine/spermine N-acetyltransferase13,14 (the enzyme in-
volved in the catabolism and salvage pathways of
spermine and spermidine), that they may inhibit the
uptake of the natural polyamines by the cells,15 and
that, as a result, they deplete endogenous polyamine
pools needed for cell replication.16 Cell death can result
from any one of these effects or from several of them
acting in tandem. On the other hand, the main advan-
by now well-established that N,ωN-bisethyl derivatives
R
of spermine and its higher and lower tetramine homo-
logues show promise as inhibitors of tumor cell prolif-
eration.2 They are increasingly mentioned in the patent
literature on anticancer drugs,3 and their pharmacologi-
cal effects have recently been exhaustively discussed.4
As is the case with most anticancer drugs, bisethyl
tetramines are not devoid of systemic toxicity and their
efficacious cytotoxic activity has to be balanced against
their toxic side effects. Therefore, the search for chemi-
cal variants of the bisethyl polyamine structures with
broader therapeutic windows is being actively pursued.
Hydroxylated polyamine derivatives have recently been
prepared in an effort to improve the toxicity profile of
the tetramines,5 conformational restrictions were in-
troduced in the polyamine backbone to enhance their
* To whom correspondence should be addressed. Phone: 608-231-
3875, ext. 12 or 23. Fax: 608-231-3892. E-mail: hsbasu@slil.com,
bjfrydman@slil.com.
10.1021/jm000309t CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/16/2000

Conformationally Restricted Analogues of BE-4-4-4
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 391
Sch em e 1
Sch em e 2
tage in the design of new tetramines is that structure-
activity studies (SARs) have shown that relatively small
structural changes in the aliphatic skeletons of the
polyamines can cause pronounced differences in their
pharmacological behavior and toxic side effects as well
as their antineoplastic activities both at the cellular
level as well as in animal models.17-19
the diamide 2 in the presence of NaH, it was possible
to secure the tetramides 3 and 6 in good yields.
Hydrolysis of the mesitylenesulfonyl protecting groups
using hydrogen bromide in glacial acetic acid in the
presence of phenol, followed by treatment of the free
base with hydrogen chloride, allowed us to obtain 4 and
7. Using a similar choice of reactions starting with the
trans- and cis-cyclobutane 1,2-bis(oxomesitylenesulfo-
nyl) esters 8 and 11 (Scheme 2), it was possible to obtain
the corresponding tetramines 10 and 13 as their hy-
drochlorides. Unsaturated cis- and trans-isomers of
bisethylhomospermine were obtained starting with the
diesters 14 and 17; on condensation with diamide 2 they
gave the tetramides 15 and 18; deprotection allowed us
to obtain 16 and 19 (Scheme 3).
All the aforementioned tetramines had cis/ trans-
geometry at the central butane segment of the molecule.
To examine the influence of planarity at this segment,
we prepared the acetylenic tetramine 22 following the
usual reaction pattern and starting with 20 (Scheme
4). In addition, the central butane segment was fixed
in the cisoid configuration by making it part of a 1,2-
benzenedimethyl structure as in 25. The synthesis of
25 followed the established reaction pattern (Scheme
4). A fully unsaturated tetramine was prepared where
three cis-double bonds where introduced in the homo-
spermine skeleton. Starting with the unsaturated dia-
mide 267 it was dimerized by reaction with the diester
17 (Scheme 5). The resulting fully unsaturated tetra-
mide was deprotected to give 28. A dienic tetramine was
prepared by alkylation of 26 with chloride 297 that gave
30; after deprotection 31 was secured (Scheme 6).
The second possible cis-diene formally derived from
homospermine was prepared by dialkylation of amide
26 with the bis(mesitylenesulfonyl) ester of 1,4-butane-
diol 32. The reaction allowed us to obtain 34 (SL-11114)
(Scheme 7). Finally, and by alkylation of amide 2 with
the iodoalkylamide 35,7 it was possible to secure the
It has been repeatedly shown that 1N,14N-bisethyl-
homospermine (BE-4-4-4), a higher homologue of bis-
ethylspermine, is a powerful cytotoxic drug but has a
narrow therapeutic window.11,20,21 The results for ani-
mal trials reported against tumors such as L1210
leukemia or Lewis lung carcinoma grafted in athymic
nude mice are indeed impressive; about a 6-fold increase
in lifespan, as compared to control, was achieved.22
However, maximum tolerated dose (MTD) values for
multiple injection (ip) schedules were found to be ca. 6
mg/kg, doses close to the levels necessary to achieve
efficient antitumor activity in human tumor xenografts.20
We, therefore, decided to apply the principle of confor-
mational restriction to the BE-4-4-4 structure, prompted
by the promising results obtained with conformationally
restricted analogues of bisethylspermine.6 Since the
latter was found to be highly cytotoxic against a line of
human prostate cancer cells,6 we assayed the new
tetramines against several lines of human prostate
cancer cells: LnCap, DU 145, DuPro, and PC-3. Pre-
liminary results suggest that it is possible to markedly
improve the therapeutic efficacy of BE-4-4-4-like com-
pounds against human prostate cancer cells by certain
conformational restrictions.
Ch em istr y
The synthesis of the trans- and cis-cyclopropane
derivatives of bisethylhomospermine made use of the
trans- and cis-cyclopropane 1,2-bis(oxomesitylenesulfo-
nyl) esters 1 and 5 (Scheme 1) whose synthesis we
described previously.6 By condensation of 1 and 5 with

392 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Valasinas et al.
Sch em e 3
Sch em e 4
Sch em e 5
Sch em e 6
Sch em e 7
Sch em e 8
second possible cis-monounsaturated isomer of BE-4-
4-4, namely, 37 (Scheme 8). The new synthetic tetra-
mines allowed us to revisit the cytotoxic behavior of the
higher homologues of bisethylspermine in the search for
more efficacious and less toxic drugs.
mines. LnCaP, DU145, and DuPro were more sensitive
than was PC-3 to all but one tetramine (25); 25 was
inactive and 22 was less active in all four cell lines. Ten
out of 12 new tetramines had ID50 values less than 1
µM in all but PC-3 cells, and several of them had ID50
values in the range 10-100 nM. PC-3 cells are in
general more resistant to the polyamine analogues than
are the other three cell lines, possibly because of
decreased polyamine transport in these cells.
We selected the DuPro cell line that exhibits an
intermediate level of sensitivity to the tetramines (Table
1) as a representative cell line for further studies. All
new tetramines plus BE-4-4-4 at 1 and 10 µM were
assayed for their effects on changes in cell number and
cellular polyamine levels over time. The effects of the
Biologica l Resu lts a n d Discu ssion
The concentration of each new tetramine required to
inhibit 50% cell growth (ID50) on day 6 of treatment was
determined by a MTT assay. The ID50 values for each
tetramine in the human prostate cancer cell lines
LnCap, DU145, DuPro, and PC-3 along with their SLIL
identification numbers and chemical structures are
shown in Table 1. Each cell line showed a slightly
different sensitivity when treated with the new tetra-

Conformationally Restricted Analogues of BE-4-4-4
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 393
Ta ble 1. Effect of Polyamine Analogues on Human Prostate Tumor Cell Growth by MTT Assay
tetramines on DuPro cell growth along with the cellular
polyamine levels are shown in Figures 1-6. At 1 µM,
most tetramines were ineffective in growth inhibition
(Figures 1-3). Only 31 and 25 inhibited cell growth by
approximately 50% at 1 µM (Figure 3A). At 10 µM,
however, all but two tetramines exhibited significant
growth inhibition (Figures 4-6). The exceptions were
22 and 25 that showed almost no detectable growth
inhibition (Figure 4A). One tetramine 37 was better
than BE-4-4-4 in inhibiting cell growth (Figure 6A). At
1 µM, none of the tetramines were able to deplete
cellular polyamine pools even on day 6 after treatment
(Figures 1-3). At 10 µM, most tetramines except 25,
markedly depleted cellular putrescine and spermidine
levels within day 4 of treatment without appreciably
changing the cellular spermine level even by day 6 of
treatment (Figures 4-6). The abilities of various tetra-
mines to deplete cellular putrescine and spermidine
levels, however, could not be correlated with their
growth inhibitory effects. For example, 10 at 10 µM had
much less effect on cellular putrescine and spermidine
pools as compared to five other tetramines: BE-4-4-4,
4, 7, 16, and 19 (Figures 4B,C and 5B,C). However 10
inhibited cell growth to the same extent as did the other
five tetramines (Figures 4 and 5). On the other hand,
on day 6 of treatment with 10 µM, 22 depleted the
polyamine pool to the same extent as did 10. However,
22 had no observable effect on cell growth, while 10
showed marked growth inhibition (Figures 4A and 5A).
Variations in intracellular tetramine levels were also
observed; 25 was poorly taken up by the cells and, as
mentioned above, exhibited no cell growth inhibition
(Figures 1 and 4). Except for this tetramine, however,
no reasonable correlation could be established between
intracellular tetramine levels and their growth inhibi-
tory activities. For example, on day 6 of treatment at
10 µM, the intracellular level of 13 was 10-fold higher
than that of 10, while both were equally efficient in

394 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Valasinas et al.
F igu r e 1. (A) Effects of 1 µM BE-4-4-4, 16, 19, 22, and 25 on the growth of DuPro cells. Symbol for each tetramine is shown in
the inset. Each data point is an average of at least three separate experiments run in duplicate. Error bars where not shown are
smaller than the symbol size. (B) Effects of 1 µM BE-4-4-4, 16, 19, 22, and 25 on the polyamine levels of DuPro cells on day 4 of
treatment. Symbol for each polyamine is shown in the inset. Each data point is an average of two separate experiments. (C)
Effects of 1 µM BE-4-4-4, 16, 19, 22, and 25 on the polyamine levels of DuPro cells on day 6 of treatment. Symbol for each
polyamine is shown in the inset. Each data point is an average of two separate experiments.
inhibiting cell growth (Figure 5). On the other hand,
on day 6 of 10 µM treatments, the intracellular level of
10 was similar to that of 22, while 10 was a much better
growth inhibitor than was 22 (compare Figures 4 and
5). It is evident that in most cases, the growth inhibitory
activities of the tetramines cannot be correlated either
to their intracellular levels and/or to their abilities to
deplete intracellular polyamine pools.
Cytotoxicities of 10 active tetramines were further
studied by examining their effects on the colony-forming
efficiency (CFE) of cells on day 6 of treatment. The
results are shown in Figures 7 and 8. Cytotoxicity of
BE-4-4-4 is included in both figures for comparison. cis-
Dehydro-BE-4-4-4 (19) was as cytotoxic as BE-4-4-4. At
10 µM, it was 15-fold more cytotoxic than the trans-
isomer 16 (Figure 7). When the cis-double bond was
placed at the outer segment of the molecule (as in 37),
cytotoxicity was greatly enhanced and the resultant
tetramine was 1 order of magnitude more cytotoxic than
the saturated tetramine BE-4-4-4 (Figure 8). Introduc-
tion of additional double bonds, as in 34 and 31, reduced
cytotoxicity. These results parallel those observed for
unsaturated pentamines, and a possible rationale for
these changes in cytotoxicities has previously been
discussed.7
Restricting rotational freedom of the central butane
chain by the introduction of alicyclic groups, however,
increased the cytotoxicity of the tetramines. Three of
the four tetramines containing alicyclic groups (4, 7, and
10) at 10 µM concentrations were 10-50-fold more

Conformationally Restricted Analogues of BE-4-4-4
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 395
F igu r e 2. (A) Effects of 1 µM BE-4-4-4, 4, 7, 10, and 13 on the growth of DuPro cells. Symbol for each tetramine is shown in the
inset. Each data point is an average of at least three separate experiments run in duplicate. Error bars where not shown are
smaller than the symbol size. (B) Effects of 1 µM BE-4-4-4, 4, 7, 10, and 13 on the polyamine levels of DuPro cells on day 4 of
treatment. Symbol for each polyamine is shown in the inset. Each data point is an average of two separate experiments. (C)
Effects of 1 µM BE-4-4-4, 4, 7, 10, and 13 on the polyamine levels of DuPro cells on day 6 of treatment. Symbol for each polyamine
is shown in the inset. Each data point is an average of two separate experiments.
cytotoxic than was BE-4-4-4 (Figures 7 and 8). The
tetramines with cyclopropyl groups were more cytotoxic
than were the tetramines with cyclobutyl groups. Al-
though no significant difference was observed between
the cytotoxicity of the cis- and trans-cyclopropyl isomers
4 and 7 (Figure 7), for the cyclobutyl isomers, the trans-
isomer 10 was approximately 20-fold more cytotoxic
than was the cis-isomer 13 (Figure 7). These differences
in cytotoxicities of the isomeric pairs 16/19 and 10/13
were not detected in the cell growth inhibition assays
(Figures 1-6). This could be due to the shorter time
span of the cell growth assay (6 days) relative to the
CFE assay (14-16 days). The cis-cyclobutyl isomer 13
is in that conformation where the pharmacophores, i.e.
the 1,2-NH2+R chains, are in an eclipsed conformation.
Therefore, this conformer is at a higher energy state
than is the trans-isomer 10, where the pharmacophores
are in a staggered conformation. The lower cytotoxicity
of the former could result from this unfavorable steric
effect that in turn may affect the compound’s interac-
tions with DNA and other cellular macromolecules. We
have now initiated a detailed nucleic acid binding study
of these tetramines to better understand this effect.
Interestingly, the presence of an alicyclic ring reduced
the systemic toxicity of the tetramines; thus, 4, 7, and
10 displayed 10-fold higher host tolerance than did BE-
4-4-4 (C. Bacchi, personal communication). Therefore,
conformational restriction by alicyclic groups enhances
cytotoxicity while lowering systemic toxicity, thus wid-

396 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Valasinas et al.
F igu r e 3. (A) Effects of 1 µM BE-4-4-4, 28, 34, 37, and 31 on the growth of DuPro cells. Each data point is an average of at least
three separate experiments run in duplicate. Symbol for each tetramine is shown in the inset. Error bars where not shown are
smaller than the symbol size. (B) Effects of 1 µM BE-4-4-4, 28, 34, 37, and 31 on the polyamine levels of DuPro cells on day 4 of
treatment. Symbol for each polyamine is shown in the inset. Each data point is an average of two separate experiments. (C)
Effects of 1 µM BE-4-4-4, 28, 34, 37, and 31 on the polyamine levels of DuPro cells on day 6 of treatment. Symbol for each
polyamine is shown in the inset. Each data point is an average of two separate experiments.
ening the therapeutic window of the new derivatives
(Frydman et al., unpublished results).
icity of the trans-isomer 10 goes above that of BE-4-4-
4. When a cyclopentyl ring is introduced, an inactive
tetramine is obtained (data not shown). To the best of
our knowledge, the bisethyl trans-cyclopropyl tetramine
4 is the most cytotoxic polyamine analogue yet reported
in human prostate tumor cell lines. These results fall
in line with a well-known tenet of drug design. When a
rigid analogue of a flexible drug molecule is constructed
by adding atoms and/or bonds to the original molecule,
the rigid analogue should be as close to the parent
molecule in size and shape as possible to be biologically
active.
Con clu sion s
The most revealing conclusion to be drawn from the
data reported above is that by introducing conforma-
tional restrictions in the otherwise freely rotating BE-
4-4-4 structure, it is possible to enhance the cytotoxic
effect of the tetramine. The growth inhibitory activity
is highly enhanced by the introduction of a cyclopropyl
ring into the central segment of the BE-4-4-4 chain.
Both the cis-isomer 4 and trans-isomer 7 of the cyclo-
propyl tetramine are highly cytotoxic. When the larger
cyclobutyl ring is introduced, cytotoxicity of the cis-
isomer 13 drops below that of BE-4-4-4 while cytotox-
Cytotoxicity of the BE-4-4-4 molecule was also en-
hanced by introducing a cis-double bond in the outer
segment of the tetramine (as in 37). This supports our

Conformationally Restricted Analogues of BE-4-4-4
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 397
F igu r e 4. (A) Effects of 10 µM BE-4-4-4, 16, 19, 22, and 25 on the growth of DuPro cells. Symbol for each tetramine is shown
in the inset. Each data point is an average of at least three separate experiments run in duplicate. Error bars where not shown
are smaller than the symbol size. (B) Effects of 10 µM BE-4-4-4, 16, 19, 22, and 25 on the polyamine levels of DuPro cells on day
4 of treatment. Symbol for each polyamine is shown in the inset. Each data point is an average of two separate experiments. (C)
Effects of 10 µM BE-4-4-4, 16, 19, 22, and 25 on the polyamine levels of DuPro cells on day 6 of treatment. Each data point is an
average of two separate experiments.
previous suggestions that a cisoid bending at one
extreme of the polyamine molecule will favor its inter-
action with DNA and increase its cytotoxicity by dis-
placing natural polyamines from their DNA binding
site(s).9 If the central butane segment is made very rigid
by introducing two double bonds, as in 34 and 31, or a
triple bond, as in 4, the growth inhibitory activity is
either reduced or completely abolished. Introduction of
a 1,2-benzenedimethyl residue, as in 25, made the
compound unacceptable for cellular uptake. The lack of
correlation for the new tetramines between the deple-
tion of the endogenous cellular polyamine pools or the
cellular uptake of the most of the tetramines and their
cytostatic effects suggests that the latter is a result of
their specific structural features. This again points in
the direction of their specific binding to larger molecules.
The enhanced host tolerance to the alicyclic tetramines
is undoubtedly related to pharmacokinetic and meta-
bolic factors that are currently under investigation.
Exp er im en ta l Section
NMR spectra were obtained using a Bruker AM-250 spec-
trometer. Reactions were monitored using TLC on silica gel
plates (0.25 mm thick). Flash chromatography was performed
on columns packed with EM Science silica gel, 230-400 mesh
ASTM. Melting points were determined on an Electrothermal
IA 9100 digital melting point apparatus. Mass spectra (ESI)
were obtained on a PE Sciex API 365 electrospray triple
quadrupole spectrometer; matrix-assisted laser desorption
ionization (MALDI) mass spectra were obtained using a
Bruker Biflex III spectrometer operating in the time-of-flight

398 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Valasinas et al.
F igu r e 5. (A) Effects of 10 µM BE-4-4-4, 4, 7, 10, and 13 on the growth of DuPro cells. Symbol for each tetramine is shown in
the inset. Each data point is an average of at least three separate experiments run in duplicate. Error bars where not shown are
smaller than the symbol size. (B) Effects of 10 µM BE-4-4-4, 4, 7, 10, and 13 on the polyamine levels of DuPro cells on day 4 of
treatment. Symbol for each polyamine is shown in the inset. Each data point is an average of two separate experiments. (C)
Effects of 10 µM BE-4-4-4, 4, 7, 10, and 13 on the polyamine levels of DuPro cells on day 6 of treatment. Symbol for each polyamine
is shown in the inset. Each data point is an average of two separate experiments.
mode; mass spectra (FAB) were obtained on an Autospec (VG)
spectrometer. HPLC analyses of the dansyl derivatives of the
polyamine tetramines were routinely performed to check the
purity of the samples. A Vydac C-18 (300-µm pore) column for
separations and a fluorescence spectrometer (340-nm excita-
tion, 515-nm emission) for detection were used.
3,8,13,18-Tet r a k is(m esit ylen esu lfon yl)-10,11-[(E)-1,2-
cyclop r op yl]-3,8,13,18-tetr a a za eicosa n e (3). Amide 223 (2.0
g, 4.16 mmol) and diester 16 (0.92 g, 1.98 mmol) were dissolved
in 40 mL of anhydrous DMF under argon, the solution was
cooled to 5 °C, and NaH (85%, 0.13 g) was slowly added with
continuous stirring. After 4 h the solid disappeared, the
reaction was then quenched with water (5 mL), the solution
was evaporated to dryness and the resulting slurry was
partitioned between ethyl acetate (20 mL) and water (3 × 15
mL). The organic layer was evaporated and the residue
purified by flash chromatography using hexane-ethyl acetate
(4:1) as eluant; 1.2 g (58%) of 3 was obtained: mp 55-56 °C;
1H NMR (CDCl3) δ 0.04-1.13 (m, 2H), 0.74 (m, 2H), 0.98 (t,
6H), 1.37 (m, 8 H), 2.29 (s, 12H), 2.56 (s, 24H), 2.81 (m, 2H),
3.13 (m, 14H), 6.93 (s, 8H); 13C NMR (CDCl3) δ 2.72,13.82,
20.93, 22.75, 24.55, 24.80, 40.04, 44.53, 44.90, 45.22, 131.90,
131.96, 133.15, 133.43, 140.04, 140.11, 142.23, 142.44; MS-
FAB (m/z) calcd 1027.46 (M+), found 1027.50.
3,8,13,18-T e t r a a z a -10,11-[(E )-1,2-c y c lo p r o p y l]e i -
cosa n e Tetr a h yd r och lor id e (4). Phenol (1.94 g, 20.66 mmol)
and 33% hydrogen bromide in glacial acetic acid (9.9 mL) were
added in tandem to solution of 3 (0.53 g, 0.52 mmol) in 6 mL
of methylene chloride at 25 °C. Stirring was kept on for 18 h,
water (5 mL) was then added, the aqueous layer separated,
reextracted with methylene chloride (3 × 8 mL), and the
aqueous solution was evaporated to dryness. The residue was
dissolved in 10 N sodium hydroxide (3 mL), the solution
extracted with chloroform (10 × 6 mL), the pooled organic

Conformationally Restricted Analogues of BE-4-4-4
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 399
F igu r e 6. (A) Effects of 10 µM BE-4-4-4, 28, 34, 37, and 31 on the growth of DuPro cells. Symbol for each tetramine is shown
in the inset. Each data point is an average of at least three separate experiments run in duplicate. Error bars where not shown
are smaller than the symbol size. (B) Effects of 10 µM BE-4-4-4, 28, 34, 37, and 31 on the polyamine levels of DuPro cells on day
4 of treatment. Symbol for each polyamine is shown in the inset. Each data point is an average of two separate experiments. (C)
Effects of 10 µM BE-4-4-4, 28, 34, 37, and 31 on the polyamine levels of DuPro cells on day 6 of treatment. Symbol for each
polyamine is shown in the inset. Each data point is an average of two separate experiments.
extracts were dried (Na2SO4), evaporated to dryness, the
residue dissolved in dry ether, and hydrogen chloride was
passed through the solution kept at 5 °C. The white precipitate
was filtered and crystallized from ethanol-ether; 0.14 g (60%)
131.92, 133.15, 133.42, 139.98, 140.00, 140.04, 142.17, 142.37;
MS-FAB (m/z) calcd 1027.46 (M+), found 1027.44.
3,8,13,18-T e t r a a z a -10,11-[(Z )-1,2-c y c lo p r o p y l]e i -
cosa n e tetr a h yd r och lor id e (7) was obtained (84%) from the
tetramide 6 following the procedure described for 4: 1H NMR
(D2O) δ 0.58 (dd, 1H), 1.17 (m, 1H), 1.29 (t, 6H), 1.41 (m, 2H),
1.80 (m, 8H), 2.91 (m, 2H), 3.12 (m, 12H), 3.42 (m, 2H); 13C
NMR (D2O) δ 12.46, 13.36, 14.95, 25.68, 45.73, 49.10, 49.48,
50.15; MS-ESI (m/z) calcd 299.52 (M+ + 1), found 299.55. Anal.
(C17H42Cl4N4) C, H, N.
1
of 4 was obtained: mp 250 °C dec; H NMR (D2O) δ 0.57 (m,
1H), 1.14 (m, 1H), 1.29 (6H), 1.40 (m, 2H), 1.80 (m, 8H), 2.92
(m, 2H), 3.12 (m, 12H), 3.41 (m, 2H); 13C NMR (D2O) δ 12.47,
13.31, 14.92, 25.61, 45.69, 49.04, 49.44, 50.12; MS-ESI (m/z)
calcd 299.52 (M+ + 1), found 299.54. Anal. (C17H42Cl4N4) C,
H, N.
3,8,13,18-Tet r a k is(m esit ylen esu lfon yl)-10,11-[(Z)-1,2-
cyclop r op yl]-3,8,13,18-tetr a a za eicosa n e (6) was prepared
(71%) following the procedure described for 3: mp 56-58 °C;
1H NMR (CDCl3) δ 0.02 (m, 1H), 0.75 (m, 2H), 0.98 (t, 6H),
1.36 (m, 8H), 2.28 (s, 12H), 2.56 (s, 2H), 2.82 (m, 4H), 3.11 (m,
12H), 6.93 (s, 8H); 13C NMR (CDCl3) δ 12.69, 13.82, 20.86,
22.66, 22.83, 24.53, 24.77, 40.03, 44.44, 44.89, 45.20, 131.85,
3,8,13,18-Tetr a k is(m esitylen esu lfon a m id o)-10,11-[(E)-
1,2-cyclobu tyl]-3,8,13,18-tetr a a za eicosa n e (9). Diester 86
(3.0 g, 6.25 mmol) and diamide 2 (6.0 g, 12.5 mmol) were
dissolved in dry DMF (50 mL), the solution was stirred and
kept at 5 °C during 2 h while NaH (0.6 g) was added and then
further stirred at 25 °C during 18 h. The solution was then
evaporated to dryness, the residue dissolved in chloroform, the

400 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Valasinas et al.
3,8,13,18-Tetr a k is(m esitylen esu lfon a m id o)-10,11-[(Z)-
1,2-cyclobu tyl]-3,8,13,18-tetr a a za eicosa n e (12) was pre-
pared by following the procedure described for 9. From 2.0 g
of the diester 116 (4.1 mmol) and 4.0 g (8.2 mmol) of diamide
2, 3.4 g (80%) of tetramide 12 was obtained: 1H NMR (CDCl3)
δ 0.98 (t, 6H), 1.40 (m, 10H), 1.80 (m, 2H), 2.30 (m, 14H), 2.55
(s, 24H), 3.15 (m, 14H), 6.95 (s, 8H); 13C NMR (CDCl3) δ 12.62,
20.79, 22.70, 24.38, 26.55, 33.85, 39.93, 41.88, 44.31, 45.06,
45.18, 131.78, 132.96, 138.82, 139.80, 141.96, 142.33; MS-FAB
(m/z) calcd 1064.48 (M++ Na), found 1064.49.
3,8,13,18-Tetr a a za -10,11-[(Z)-1,2-cyclobu tyl]-3,8,13,18-
eicosa n e tetr a h yd r och lor id e (13) was obtained (85%) fol-
lowing the procedure described for 10: 1H NMR (D2O) δ 1.30
(t, 6H), 1,80 (m, 10H), 2.20 (m, 2H), 2.90 (m 2H), 3.10 (m, 16H);
13C NMR (D2O) δ 13.29, 25.22, 25.56, 35.59, 45.66, 49.62, 50.67,
53.94; MS-ESI (m/z) calcd 313.54 (M+ + 1), found 313.56. Anal.
(C18H44Cl4N4) C, H, N.
(E)-3,8,13,18-Tet r a k is(m esit ylen esu lfon yl)-3,8,13,18-
tetr a a za eicos-10-en e (15) was obtained (56%) by condensa-
tion of the diester 146 and the diamide 2 following the
procedure described for 3: 1H NMR (CDCl3) δ 0.95 (t, J ) 7.1
Hz, 6H), 1.34 (m, 8H), 2.29 (s, 24H), 3.09 (m, 12H), 3.72 (d, J
) 4.5 Hz, 4H), 5.48 (t, J ) 4.3 Hz, 2H), 6.92 (s, 4H), 6.93 (s,
4H); 13C NMR (CDCl3) δ 12.71,20.90, 22.71, 22.76, 24.74, 40.04,
42.21, 44.56, 45.69, 128.45, 131.88, 132.02, 140.16, 142.20,
142.58; MS-FAB (m/z) calcd 1013.44 (M+), found 1013.42.
(E)-3,8,13,18-Tetr a a za eicos-10-en e tetr a h yd r och lor id e
(16) was obtained (75%) from 15 following the procedure
described for 4: mp >230 °C; 1H NMR (D2O) δ 1.26 (t, J )
12.5 Hz, 6H), 1.79 (m, 8H), 3.12 (m, 12H), 3.80 (d, J ) 7.2,
4H), 6.10 (m, 2H); 13C NMR (D2O) δ 12.79, 25.10, 45.19, 48.53,
48.62, 50.36, 130.66; MS-ESI (m/z) calcd 285.49 (M+ + 1), found
285.50. Anal. (C16H40Cl4N4) C, H, N.
F igu r e 7. Effects of increasing concentration of BE-4-4-4 (b),
4 (1), 7 (b), 10 ([), 13 (.), 16 (9), and 19 (2) on the survival
of DuPro cells by CFE assay. Each data point and correspond-
ing error bars are, respectively, an average and the standard
deviation of six independent observations.
(Z)-3,8,13,18-Tet r a k is(m esit ylen esu lfon yl)-3,8,13,18-
tetr a a za eicos-10-en e (18) was obtained (55%) by condensa-
tion of diester 176 with the diamide 2 following the procedure
described for 3: 1H NMR (CDCl3) δ 0.96 (t, 6H), 1.28 (br, 8H),
2.29 (s, 12H), 2.56 (s, 24H), 3.09 (m, 12H), 3.72 (d, 4H), 5.49
(br, 2H), 6.92 (s, 4H), 6.93 (s, 4H); 13C NMR (CDCl3) δ 12.66,
20.89, 22.71, 24.67, 39.97, 42.13, 44.67, 45.60, 128.40, 131.85,
131.97, 133.00, 133.50, 140.14, 142.30, 142.54; MS-FAB (m/z)
calcd 1013.44 (M+), 1013.45.
(Z)-3,8,13,18-Tetr a a za eicos-10-en e tetr a h yd r och lor id e
(19) was obtained (74%) from 18 following the procedure
1
described for 4: H NMR (D2O) δ 1.29 (t, 6H), 1.79 (m, 8H),
3.12 (m, 12H), 3.84 (d, 4H), 5.96 (t, J ) 4.6 Hz, 2H); 13C NMR
(D2O) δ 12.79, 25.09, 25.23, 45.19, 46.27, 48.54, 48.86, 128.68;
MS-ESI (m/z) calcd 285.49 (M+ + 1), found 285.49. Anal.
(C16H40Cl4N4) C, H, N.
3,8,13,18-Tet r a k is(m esit ylen esu lfon yl)-3,8,13,18-t et -
r a a za eicos-10-yn e (21) was obtained (59%) by condensation
of diester 206 with amide 2 as described for 3: 1H NMR (CDCl3)
δ 0.90 (t, 6H), 1.30 (bs, 8H), 2.20 (s, 12H), 2.45 (s, 24H), 3.05
(m, 12H), 3.75 (s, 4H), 6.87 (s, 8H); 13C NMR (CDCl3) δ 12.70,
20.78, 22.68, 34.65, 39.97, 44.46, 44.99, 78.62, 131.85, 131.98,
132.34, 140.14, 142.13, 142.55; MS-FAB (m/z) calcd 1011.12
(M+), found 1011.10.
3,8,13,18-Tetr a a za eicos-10-yn e tetr a h yd r och lor id e (22)
was obtained (76%) from 21 following the procedure described
for 4: mp > 280 °C; 1H NMR (D2O) δ 1.30 (t, 6H), 1.80 (b,
8H), 2.90-3.25 (m, 12H), 4.05 (s, 4H); 13C NMR (D2O) δ 13.39,
25.64, 39.26, 45.72, 49.20, 81.20; MS-ESI (m/z) calcd 283.47
(M+ + 1), found 283.45. Anal. (C16H38Cl4N4) C, H, N.
10,11-Ben zo-3,8,13,18-tetr a kis(m esitylen esu lfon yl)-3,8,-
13,18-tetr a a za eicosa n e (24) was obtained (84%) by conden-
sation of diester 236 with amide 2 as described for 3: mp 109
°C (from ethyl acetate-hexane); 1H NMR (CDCl3) δ 0.93 (t,
6H), 1.20 (m, 8H), 2.30 (s, s, 12H), 2.65-2.70 (s, s, 24H), 2.90
(m, 8H), 3.10 (t, 6H), 4.25 (s, 4H), 6.90, 6.95 (s, s, 8H), 7.25 (s,
4H); 13C NMR (CDCl3) δ 12.66, 20.84, 22.67, 23.12, 24.71,
39.80, 40.06, 44.32, 44.46, 45.30, 47.28, 128.05, 129.56, 131.78,
132.36, 133.19, 134.27, 139.89, 142.14, 151.27; MS-FAB (m/z)
calcd 1063.50 (M+), found 1063.49.
F igu r e 8. Effects of increasing concentration of BE-4-4-4 (b),
34 (2), 37 (1), and 31 ([) on the survival of DuPro cells by
CFE assay. Each data point and corresponding error bars are,
respectively, an average and the standard deviation of six
independent observations.
organic layer washed first with water, then with concentrated
ammonium chloride, dried (Na2SO4) and evaporated to dryness.
The residue was purified by flash chromatography using
hexane-ethyl acetate (from 8:2 to 7:3) as eluant; 5.7 g (87%)
of 9 was obtained as a glassy oil: 1H NMR (CDCl3) δ 1.05 (t,
6H), 1.30 (m, 10H), 1.80 (m, 2H), 2.15 (m, 2H), 2.35 (s, 12H),
2.52 (s, 24H), 3.10 (m, 12h), 3.35 (m, 2H), 6.95 (s, 8H); 13C
NMR (CDCl3) δ 12.75, 20.89, 22.70, 23.35, 24.38, 24.65, 24.78,
37.62, 40.10, 44.61, 45.40, 50.25, 131.87, 133.51, 139.96,
142.16; MS-FAB (m/z) calcd 1064.48 (M+ + Na), found 1064.45.
3,8,13,18-Tetr a a za -10,11-[(E)-1,2-cyclobu tyl]-3,8,13,18-
eicosa n e tetr a h yd r och lor id e (10) was obtained (80%) from
9 following the procedure described for 4; 5.7 g of 9 gave 2.0 g
1
of 10: mp > 300 °C dec; H NMR (D2O) δ 1.30 (t, 6H), 1.80
(m, 10H), 2.15 (m, 2H), 2.50 (m, 2H), 3.15 (m, 16H); 13C NMR
(D2O) δ 13.28, 13.36, 25.48, 25.58, 37.81, 45.66, 49.03, 49.82,
53.94; MS-ESI (m/z) calcd 313.54 (M+ + 1), found 313.56. Anal.
(C18H44Cl4N4) C, H, N.
10,11-Ben zo-3,8,13,18-t et r a a za eicosa n e t et r a h yd r o-

Conformationally Restricted Analogues of BE-4-4-4
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 401
ch lor id e (25) was obtained (56%) from 24 following the
procedure described for 4: mp > 300 °C dec; 1H NMR (D2O) δ
1.25 (t, 6H), 1.75 (m, 8H), 3.00-3.30 (m, 12H), 4.45 (s, 4H),
7.60 (s, 4H); 13C NMR (D2O) δ 13.40, 25.74, 25.83, 45.73,49.11,
49.77, 50.72, 133.32, 134.15; MS-ESI (m/z) calcd 335.55 (M+
+ 1), found 335.56. Anal. (C20H42Cl4N4) C, H, N.
(Z,Z,Z)-3,8,13,18-Tetr a k is(m esitylen esu lfon yl)-3,8,13,-
18-tetr a a za eicosa -5,10,15-tr ien e (27) was prepared by con-
densation of diamide 267 (2.4 g, 5 mmol) with diester 17 (1.07
g, 2.37 mmol) in the presence of 0.155 g of NaH (85%) following
the procedure described for 3. After chromatography 2.5 g
(49%) of 27 was obtained: 13C NMR (CDCl3) δ 12.84, 20.93,
22.69, 40.45, 41.49, 42.17, 128.02, 128.58, 129.14, 131.93,
132.06, 140.19, 140.32, 142.24, 142.45; MS-FAB (m/z) calcd
1009.41 (M+), found 1009.42.
described for 4: 1H NMR (D2O) δ 1.29 (t, 3H), 1.31 (t, 3H),
1.79 (m, 8H), 3.12 (m, 12H), 3.83 (m, 4H), 5.96 (t, 2H); 13C
NMR (D2O) δ 13.33, 13.43, 25.64, 25.78, 45.37, 45.71, 46.19,
46.77, 49.08, 49.34, 49.69, 129.20, 129.34; MS-ESI (m/z) calcd
285.49 (M+ + 1), found 285.50. Anal. (C16H40Cl4N4) C, H, N.
1,4-Bis(m esitylen esu lfon yloxy)bu ta n e (32). 1,4-Butane-
diol (4.5 g, 50 mmol) and benzyltriethylammonium bromide
(0.67 g, 2.5 mmol) were dissolved in a mixture of 45 mL of
50% KOH and 30 mL dioxane. The solution was kept at 5 °C
while a solution of 26 g (120 mmol) mesitylenesulfonyl chloride
in 40 mL dioxane was added in small portions. After complet-
ing the addition, stirring was kept for 4 h at 5 °C, water (200
mL) was added and the mixture further stirred at 25 °C for
18 h. The precipitate was filtered, dried, and crystallized from
ethyl acetate-hexane: 14.6 g (64%); mp 108 °C; 1H NMR
(CDCl3) δ 1.75 (m, 4H), 2.30 (s, 6H), 2.55 (s, 12H), 3.95 (m,
4H), 7.02 (s, 4H); 13C NMR (CDCl3) δ 20.86, 22.39, 25.11, 68.26,
130.50, 131.65, 139.64, 143.22; MS-MALDI (m/z) calcd 477.59
(M+ + Na), found 477.60.
Biology. Ma ter ia ls: DuPro cells were obtained from M.
Eileen Dolan of the University of Chicago, Chicago, IL. All
other cell lines used in this study were obtained from the
American Type Culture Collection (Rockville, MD). Tissue
culture medium was obtained from Fisher Scientific (Itasca,
IL) and fetal bovine serum was obtained from Gemini Bio-
products, Inc. (Calabasas, CA). All other reagents were
analytical grade. Deionized double-distilled water was used
in all studies.
Tissu e cu ltu r e:17 Cells were seeded into 75-cm2 culture
flasks with 15 mL of Eagle’s minimal essential medium
supplemented with 10% fetal calf serum and nonessential
amino acids. The flasks were incubated in a humidified 95%
air/5% CO2 atmosphere. The cells were grown for at least 24
h to ensure that they were in the log phase of growth. They
were then treated with the tetramines. Cells were harvested
by treatment for 5 min with STV (saline A, 0.05% trypsin,
0.02% EDTA) at 37 °C. The flasks were rapped on a lab bench,
pipetted several times and aliquots of the cell suspension were
withdrawn and counted using a Coulter particle counter that
was standardized for each cell line using a hemacytometer.
P olya m in e a n a lysis:24 Approximately 1 × 106 cells were
taken from harvested samples and centrifuged at 1000 rpm
at 4 °C for 5 min. The cells were washed twice by resuspending
them in chilled Dulbecco’s isotonic phosphate buffer (pH 7.4)
and centrifuged at 1000 rpm at 4 °C. The supernatant was
decanted and 250 µL of 2% perchloric acid was added to the
cell pellet. The cells were then sonicated and the lysates were
kept at 4 °C for at least 1 h. After centrifugation at 8000g
for 5 min, the supernatant was removed for analysis. An
appropriate volume of the supernatant (50-100 µL) was
fluorescence-labeled by derivatization with dansyl chloride
following procedures published elsewhere.24 Each sample
was loaded onto a C-18 high-performance liquid chromatog-
raphy column and separated at the analytical laboratory of
the University of Wisconsin Comprehensive Cancer Center
(UWCCC) using a previously published procedure.24 Peaks
were detected and quantitated using a Shimadzu HPLC
fluorescence monitor coupled to a Spectra-Physics peak inte-
grator. Because polyamine levels vary with environmental
conditions, control cultures were sampled for each experiment.
(Z,Z,Z)-3,8,13,18-Tetr a eicosa -5,10,15-tr ien e tetr a h yd r o-
ch lor id e (28) was obtained from 27 following the procedure
described for 4. From 1.45 g of 27, 0.44 g of 28 was obtained
(61%): mp 240 °C; 1H NMR (D2O) δ 1.31 (J ) 7.3 Hz, 6H),
3.14 (q, J ) 7.3 Hz, 4H), 3.85 (m, 12H), 5.97 (m, 6H); 13C NMR
(D2O) δ 13.45, 45.42, 46.23, 46.43, 129.15, 129.44, 129.57; MS-
ESI (m/z) calcd 281.46 (M+ + 1), found 281.44. Anal. (C16H36
Cl4N4) C, H, N.
-
(Z,Z)-3,8,13,18-Tetr a k is(m esitylen esu lfon yl)-3,8,13,18-
tetr a a za eicosa -5,10-d ien e (30) was prepared by condensa-
tion of the diamide 297 (0.68 g, 1.2 mmol) with the diamide
26 (0.57 g, 1.2 mmol) in the presence of NaH (85%, 90 mg)
following the procedure described for 3. After chromatography
0.95 g (79%) of 30 was obtained: 1H NMR (CDCl3) δ 0.96 (t,
3H), 0.99 (t, 3H), 1.34 (m, 4H), 2.29 (s, 12H), 2.55 (s, 24H),
3.10 (m, 8H), 3.70 (m, 8H), 5.49 (m, 4H), 6.92 (s, 8H); 13C NMR
(CDCl3) δ 12.67, 12.84, 20.90, 22.67, 22.70, 22.74, 24.69, 39.99,
40.44, 41.49, 42.09, 42.19, 42.28, 44.48, 45.54, 127.98, 128.21,
128.69, 129.20, 131.86, 131.93, 132.00, 132.05, 132.34, 140.02,
140.14, 140.17, 140.29, 142.18, 142.49, 142.59, 142.87; MS-
FAB (m/z) calcd 1011.42 (M+), found 1011.43.
(Z,Z)-3,8,13,18-Tetr a eicosa -5,10-d ien e tetr a h yd r och lo-
r id e (31) was obtained (71%) from 30 following the procedure
described for 4: 1H NMR (D2O) δ 1.29 (t, 3H), 1.31 (t, 3H), 1.79
(m, 4H), 3.12 (m, 8H), 3.83 (m, 8H), 5.96 (m, 4H); 13C NMR
(D2O) δ 13.34, 13.44, 25.64, 25.77, 45.40, 45.72, 46.22, 46.41,
46.81, 49.07, 49.39, 129.16, 129.29, 129.38, 129.54; MS-ESI
(m/z) calcd 283.47 (M+ + 1), found 283.48. Anal. (C16H38Cl4N4)
C, H, N.
(Z,Z)-3,8,13,18-Tetr a k is(m esitylen esu lfon yl)-3,8,13,18-
tetr a a za eicosa -5,15-d ien e (33) was obtained (42%) by alky-
lation of the diamide 26 with the diester 32 following the
procedure described for the synthesis of 3: 1H NMR (CDCl3)
δ 0.99 (t, 6H), 1.35 (m, 4H), 2.29 (s, 12H), 2.55 (s, 12H), 2.57
(s, 12H), 3.10 (m, 8H), 3.71 (m, 8H), 5.46 (m, 4H), 6.93 (s, 8H);
13C NMR (CDCl3): δ 12.83, 20.91, 22.68, 22.74, 24.67, 40.47,
41.49, 42.06, 45.50, 128.15, 128.87, 131.93, 131.99, 132.80,
140.02, 140.17, 140.28, 142.47, 142.59; MS-FAB (m/z) calcd
1011.42 (M+), found 1011.44.
(Z,Z)-3,8,13,18-Tetr a eicosa -5,15-d ien e tetr a h yd r och lo-
r id e (34) was obtained (80%) from 33 following the procedure
described for 4: 1H NMR (D2O) δ 1.31 (t, 6H), 1.81 (m, 4H),
3.13 (m, 8H), 3.83 (m, 8H), 5.96 (m, 4H); 13C NMR (D2O) δ
13.43, 25.77, 45.39, 46.19, 46.78, 49.35, 129.12, 129.41; MS-
ESI (m/z) calcd 283.47 (M+ + 1), found 283.48. Anal. (C16H38
Cl4N4) C, H, N.
-
MTT a ssa y:6 Trypsinized cell suspensions were diluted to
seed a 80-µL suspension of 500 cells into each well of a 96-
well Corning microtiter plate. The plates were incubated
overnight at 37 °C in a humidified 95% air/5% CO2 atmos-
phere. 20 µL of appropriately diluted stock solutions of each
drug was added to the middle 8 columns of the microtiter
plates. Each drug concentration was run in quadruplicate. The
outer columns of the plates were used for buffer controls. Cells
were incubated with the drug for 6 days. 25 µL of a 5 mg/mL
solution of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was added to each well and the plates were
incubated at 37 °C for 4 h. Cells were then lysed by incubating
at 37 °C overnight with 100 µL of lysis buffer (500 mL of the
lysis buffer contained: 100 g lauryl sulfate (SDS), 250 mL of
(Z)-3,8,13,18-Tet r a k is(m esit ylen esu lfon yl)-3,8,13,18-
tetr a a za eicos-5-en e (36) was obtained (79%) by condensation
of the iodoalkyl diamide 357 with diamide 2 following the
procedure described for the synthesis of 3: 1H NMR (CDCl3)
δ 0.97 (t, 3H), 1.00(t, 3H), 1.43 (m, 8H), 2.29 (s, 12H), 2.54 (s,
3H), 2.56 (s, 12H), 2.57 (s, 9H), 3.07 (m, 12H), 4.12 (m, 4H),
5.48 (m, 2H), 6.93 (s, 8H); 13C NMR (CDCl3) δ 12.69, 12.70,
20.91, 22.70, 22.71, 22.76, 22.91, 24.46, 24.70, 39.98, 40.48,
41.49, 41.99, 44.49, 44.93, 45.43, 128.18, 128.90, 131.88,
131.94, 132.04, 139.98, 140.12, 140.16, 142.21, 142.29, 142.48;
MS-FAB (m/z) calcd 1013.44 (M+), found 1013.44.
(Z)-3,8,13,18-Tetr a a za eicos-5-en e tetr a h yd r och lor id e
(37) was obtained (73%) from 36 following the procedure

402 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Valasinas et al.
R. H.; Marton, L. J . A stopped-flow H-D exchange kinetic study
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tion of a polyamine analogue, 1,19-bis-(ethylamino)-5,10,15-
triazanonadecane (BE-4-4-4-4), with DNA and effect on growth,
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N,N-dimethylformamide in 2 mL of glacial acetic acid, pH 4.8).
The color developed was read at room temperature at 570 nm
in a E-max precision microplate reader (Molecular Devices
Corp., Sunnyvale, CA) and data were analyzed using manu-
facturer supplied cell survival software.
CF E a ssa y:17 All of the cell lines that were used in this
assay have previously been optimized with respect to the
necessary incubation times for observable colony formation.
Both floating and attached cells were harvested and centri-
fuged at 1000 rpm for 10 min at 4 °C. The pelleted cells were
resuspended and replated in quadruplicate at appropriate
dilution into 60-mm plastic Petri dishes. The Petri dishes were
prepared not more than 24 h in advance with 4 mL of
supplemented Eagle’s minimum essential medium containing
5-10% fetal bovine serum (standardized for each cell line).
Cells were incubated for the previously standardized number
of days in a 95% air/5% CO2 atmosphere. The plates were
stained with 0.125% crystal violet in methanol and counted.
Results are expressed as a surviving fraction of an appropriate
control.
Ack n ow led gm en t. We are grateful to Ms. Amy C.
Harms and Mr. J ames F. Brown for their expert help
in securing the mass spectra and to Ms. Kendra Tutsch
(UWCCC) for performing the HPLC analyses. These
assays were performed at the Mass Spectrometry-
Bioanalytical Facility of the Biotechnology Center of the
University of Wisconsin-Madison and the analytical
laboratory of UWCCC.
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