Cyclopropane-containing polyamine analogues are efficient growth inhibitors of a human prostate tumor xenograft in nude mice

Article abstract of DOI:10.1021/jm030175u

Polyamine analogues 7, 10, 18, 27, and 32 containing cyclopropane rings were obtained by chemical synthesis. Their antineoplastic activities were assessed against the cultured human prostate tumor cell lines DU-145, DuPro, and PC-3. Decamines 32 and 27 ex

Full text of DOI:10.1021/jm030175u

4586
J . Med. Chem. 2003, 46, 4586-4600
Cyclop r op a n e-Con ta in in g P olya m in e An a logu es Ar e Efficien t Gr ow th In h ibitor s
of a Hu m a n P r osta te Tu m or Xen ogr a ft in Nu d e Mice
Benjamin Frydman,*,† Andrei V. Blokhin,† Sara Brummel,‡ George Wilding,‡ Yulia Maxuitenko,§
Aparajita Sarkar,† Subhra Bhattacharya,† Dawn Church,† Venodhar K. Reddy,† J ohn A. Kink,†
Laurence J . Marton,† Aldonia Valasinas,† and Hirak S. Basu†
SLIL Biomedical Corp., 535 Science Drive, Suite C, Madison, Wisconsin 53711-1066; Department of Medicine, University of
Wisconsin, Madison, Wisconsin 53792; and Southern Research Institute, Birmingham, Alabama 35255-5305
Received April 15, 2003
Polyamine analogues 7, 10, 18, 27, and 32 containing cyclopropane rings were obtained by
chemical synthesis. Their antineoplastic activities were assessed against the cultured human
prostate tumor cell lines DU-145, DuPro, and PC-3. Decamines 32 and 27 exhibited variable
levels of cytotoxicity against all three cell lines, while 7, 10, and 18 were efficacious against
DU-145 and DuPro. Maximum tolerated doses (MTD) for all five compounds in a NCr-nu mouse
model were determined at dosing schedules of q1d × 5 (ip) in two cycles with a break of 10
days between cycles. Their antitumor efficacies were then tested against DU-145 tumor
xenografts in mice treated with all five agents at their respective MTDs. In addition, the
efficacies of 7 and 10 against the same tumor xenograft were assessed at doses below their
respective MTDs. In all experiments, administration began two weeks after tumor implantation.
All compounds efficiently inhibited tumor growth for up to 50 days postimplantation, with
negligible animal body weight loss. Tetramine 10 and hexamine 18 were the most efficient
among the five analogues in arresting tumor growth. Tetramine 10 containing two cyclopropane
rings had the lowest systemic toxicity as reflected in animal body weight loss. It was further
assessed at a weekly administration regimen of (q1w × 4) in two cycles with a four-week break
between the cycles. At this dosing schedule, 10 again efficiently arrested tumor growth with
negligible effect on animal body weight. Tetramine 10 also arrested the growth of large tumors
(ca. 2000 mm3) treated 66 days postimplantation. Studies on the metabolism of 10 showed
that it accumulates in tumor within 6 h after the end of administration and reached a maximum
level 72 h after cessation of dosing. Intracellular concentrations of 10 in liver and kidney were
much smaller when compared to those in the tumor when measured 72 h after cessation of
dosing. In liver and kidney, the deethyl metabolites of 10 accumulated over a 96 h period after
cessation of dosing.
In tr od u ction
that ca. 220,900 new cases of prostate cancer will be
detected in the USA and about 28 800 men will die of
this disease.7 Human prostate cancer cell lines were
found to be very sensitive to in vitro treatment with
Cancer cells accumulate the polyamines spermidine
and spermine and their diamine precursor putrescine.
Cancer-bearing animals have elevated levels of poly-
amines in their extracellular fluids.1 The role of
polyamines in cell division and the potential usefulness
of polyamine analogues as antiproliferative agents
against many tumor cell lines have been extensively
discussed.2,3 Prostate cancer shares with other cancers
the above-mentioned intracellular accumulation of
polyamines, but is different in the sense that the
prostate is by itself a uniquely rich factory of polyamine
production. The semen of healthy men contains large
amounts of spermine (ca. 3 mM) that originates mainly
from prostatic secretion.4 No other human organ has
such high polyamine concentrations.5 Therefore, target-
ing prostatic polyamines has been a tempting approach
for the therapy of prostatic carcinoma.6
R
conformationally restricted N,ωN-bisethylspermine ana-
logues, as well as with their higher tetramine and
pentamine homologues.8-10 As is the case with most
anticancer drugs, the aforementioned analogues are not
devoid of systemic toxicity, and their cytotoxicities must
be balanced against their toxic side effects.11,12 A series
of maximum tolerated dose (MTD) studies in animals
helped to identify one of the least toxic among them;
namely, SL-11093 (3,8,13,18-tetraaza-10,11-[(E)-1,2-cy-
R
clopropyl]eicosane tetrahydrochloride): a Ν,ωΝ-bisethyl
polyamine analogue that strongly inhibits the growth
of cultured human prostate tumor cell lines.9 When
assayed in nude mice xenografted with the human
prostate cancer cell line DU-145, SL-11093 showed a
strong inhibitory effect on tumor growth at doses that
had minimal systemic toxicity in the animals.13
Prostate cancer is the single most common cancer in
American males.7 During the year 2003, it is estimated
The synthesis of a cyclopropane-substituted polyamine
such as SL-11093, showing good in vivo antineoplastic
effects and minimal systemic toxicity, opened a new
vista for further synthesis of polyamine analogues. The
cyclopropane ring, due to its unusual bonding and
* To whom correspondence should be addressed. Phone: (608) 231-
3875 × 12; Fax: (608) 231-3892; E-mail: bjfrydman@slil.com.
† SLIL Biomedical Corp.
‡ University of Wisconsin.
§ Southern Research Institute.
10.1021/jm030175u CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/06/2003

Cytotoxic Cyclopropane-Containing Polyamine Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4587
Sch em e 1
inherent ring strain (275 kcal/mol), is unique among
carbocycles in both its properties and reactions.14 Sub-
stituted cyclopropanes are endowed with a large spec-
trum of biological properties, ranging from enzyme
inhibitions, to antibiotic, antiviral, antitumor, and
neurological properties.15 They, therefore, seem quite
suited for the design of novel polyamine analogues. The
design of efficacious polyamine analogues poses chal-
lenges while offering tempting advantages. The main
challenge is that the precise mechanisms by which
polyamine analogues kill tumor cells and cause systemic
toxicity are still not entirely clear.16 It has been estab-
lished that polyamines and their analogues bind to
nucleic acids and alter their conformations.17 It was also
shown that in mammalian tissues, polyamine homeo-
stasis involves a complex of several sensitive feedback
systems regulating the synthesis, degradation, and
transport of polyamines.18 Disruption of this homeosta-
sis by polyamine analogues leads to polyamine depriva-
tion and can result in cell death. Cell death can also
result from all these effects acting in tandem with the
condensation of chromatin resulting from polyamine
analogues binding to the phosphodiester bonds of DNA.19
Polyamine analogues have recently been shown to
induce the small regulatory protein antizyme, and this
antizyme-inducing potential may maximize polyamine
deprivation and growth inhibition.20 There is also the
interesting observation that high levels of intracellular
polyamines promote hyperacetylation of histones,21 and
highly acetylated histones are known to signal the start
of cell regulatory events.22 On the other hand, the main
advantage in designing new polyamine analogues is that
relatively small structural changes in the aliphatic
skeleton of the polyamines can cause pronounced dif-
ferences in their pharmacological behavior and toxic side
effects, as well as in their antineoplastic activities both
at the cellular level and in animal models.23
two cyclopropane rings (10, Scheme 1). A RN,ωN-bis-
(ethyl)hexamine substituted with two cyclopropane
rings (18, Scheme 2) was also prepared, and two RN,ωN-
bis(ethyl)decamines substituted with one cyclopropane
ring (27, Scheme 3) and with three cyclopropane rings
(32, Scheme 4) were obtained by total syntheses. The
cyclopropane-containing polyamine analogues are mix-
tures of stereoisomers. Systemic toxicities and cytotoxic
effects of the aforementioned analogues were assessed
in vivo in nude mice grafted with the human prostate
cancer DU-145 cell line. Treatment with any of these
analogues resulted in marked antiproliferative effects.
Ch em istr y
The synthesis of 7 made use of the known ethyl (E)-
2-cyanocyclopropanecarboxylate 1 as a first intermedi-
ate (Scheme 1). It was reduced with lithium borohydride
to the hydroxymethyl derivative and the latter esterified
with mesitylenesulfonyl chloride to give 2. This ester
was used to alkylate mesitylenesulfonyl ethylamide, and
3 was thus prepared. Reduction of the nitrile residue
followed by a reaction with mesitylenesulfonyl chloride
gave 4. It was brought into reaction with the mesity-
lenesulfonic diester of trans-1,2-bis(hydroxymethyl)-
cyclopropane 5 to give the tetramide 6. Cleavage of the
protecting residues allowed the synthesis of the tet-
rahydrochloride 7. Condensation of 4 with the bis-
(mesitylenesulfonyl) ester of 1,4-butanediol 8 gave the
tetramide 9. Deprotection of the amine groups gave
tetrahydrochloride 10.
The synthesis of the hexamine 18 started with the
known trans-diester 11 (Scheme 2). It was reduced to
the dialcohol 12, and the latter treated with N-bromo-
succinimide and triphenylphosphine to convert it into
the dibromomethyl derivative 13. The reaction of 13
with the known diamide 14 allowed the synthesis of the
bromomethyl derivative 15. The latter was used to
alkylate the diamide 16, a reaction that allowed the
synthesis of 17. Cleavage of the mesitylenesulfonyl
residues gave hexahydrochloride 18.
Structural variations of SL-110939 led to the synthesis
of new RN,ωN-bis(ethyl)tetramines substituted with
three cyclopropane rings (7, Scheme 1) as well as with

4588 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Frydman et al.
Sch em e 2
Sch em e 3
For the synthesis of decamine 27, the pentamide
intermediate 25 was constructed starting with the
known triamide 19 (Scheme 3). The latter was alkylated
with 4-bromobutyronitrile to give nitrile 20, the nitrile
was reduced to the amine 21 and the latter acylated
with mesitylenesulfonyl chloride to give 22. By repeat-
ing the aforementioned sequence on 22, the nitrile 23
was obtained. It was then reduced to 24, and the latter
acylated to give 25. This amide was alkylated with the
bis-mesitylenesulfonyloxy derivative 5 to give the dec-
amide 26. Cleavage of the protecting groups in 26 gave
the decahydrochloride 27.
The synthesis of 32 started with the condensation of
the bromomethyl intermediate 15 with 1,4-bis (mesity-
lenesulfonyl)putrescine 16 to give 28 (Scheme 4). In
tandem, the dibromomethyl intermediate 13 was used
to alkylate mesitylenesulfonylamide to give 29. Alky-
lation of 29 with 1,4-dibromobutane gave 30; condensa-

Cytotoxic Cyclopropane-Containing Polyamine Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4589
Sch em e 4
Sch em e 5
tion of 30 with 28 gave decamide 31. Cleavage of the
protecting residues of 31 gave decahydrochloride 32.
The synthesis of possible metabolites of 10 (see below),
namely, the monoethyl derivative 37 and the dideethyl
derivative 35, were obtained as outlined in Scheme 5.
Alkylation of 1,4-bis(mesitylenesulfonyl)putrescine 16
with 2 gave the dicyanocyclopropyl derivative 33. Re-
duction of the nitrile residues with LiAlH4 followed by
sulfonylation of the free amine residues with mesity-
lenesulfonyl chloride gave 34. Cleavage of the sulfonyl
protecting groups gave 35 tetrahydrochloride. Alkyla-
tion of 34 with 1 equiv of ethyl iodide gave 36; the
deprotection of the amino residues on 36 gave tetrahy-
drochloride 37.
as the concentration of analogue required to inhibit cell
growth by 50%. The structures of the analogues and
their ID50 values are listed in Table 1. All compounds
efficiently inhibited the growth of DuPro and DU-145
cell lines at nanomolar concentrations. While 32 and
27 were effective against all three cell lines, 7, 10, and
18 have no appreciable effect on PC-3 cell growth within
the concentration range assayed. The DU-145 cell line
was the most sensitive of the cell lines assayed. Anti-
tumor efficacies of the five synthetic polyamine ana-
logues were assessed in vivo in athymic mice grafted
with the DU-145 cell line.
Toxicity studies were carried out in male athymic
NCr-nu mice housed in microisolator cages (see Ex-
perimental Section). The compounds were dissolved in
water and administered ip daily for 5 consecutive days
(q1d × 5) in a first cycle. The animals were then rested
for 10 days, and a second cycle of treatment also
followed the q1d × 5 schedule. Body weights were
measured twice a week. Tetramine 7 at 75 mg/kg was
Biologica l Resu lts a n d Discu ssion
The growth inhibitory effects of the five cyclopropane-
containing polyamine analogues were assessed against
the human prostate tumor cell lines PC-3, DuPro, and
DU-145 using a MTT assay. The ID50 value was defined

4590 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Frydman et al.
Ta ble 1
lethal, resulting in the death of all treated mice. Lower
dosages of 50, 25, and 12.5 mg/kg were well tolerated
without animal deaths. Administration of 50 mg/kg was
accompanied by maximum average body weight losses
of 8% (2 g) and 17% (4 g) after the first and second
rounds of treatment, respectively. Administration of two
cycles of 25 mg/kg produced only 1 g (4%) body weight
loss only after the second cycle of treatment, while
administration of cycles of 12.5 mg/kg did not cause any
body weight loss. The maximum tolerated dosage (MTD)
for 7 was, therefore, 50 mg/kg for this treatment
protocol.
treatment cycle. Administration of the second treatment
cycle at 12.5 mg/kg was associated with an average 13%
body weight loss (3 g). The second treatment cycle at
6.25 mg/kg did not produce any body weight loss. A 12.5
mg/kg dose was the MTD for this protocol.
Decamine 27 was toxic during the first treatment
cycle at 25 mg/kg causing 100% lethality. At 12.5 mg/
kg, the first treatment cycle was well tolerated. The
second cycle, however, was lethal for two out of three
animals. Dosages of 6.25 mg and 3.13 mg/kg were well
tolerated, without deaths or body weight loss. 6.25 mg/
kg was the MTD for this treatment protocol.
Tetramine 10 at 75 mg/kg was also lethal, resulting
in the death of all three mice. Deaths occurred during
(on day 10) and after the end (day 15) of the second cycle
of treatment. Dosages of 50, 25, and 12.5 mg/kg were
well tolerated without deaths. The 50 mg/kg dose was
associated with maximum average body weight losses
of 8% and 17% as a result of the first and second cycles
of treatment, respectively. The lower dosages of 25 and
12.5 mg/kg caused average body weight losses of 13%
and 4%, respectively, only after the second cycle of
treatment. The 50 mg/kg dose was the MTD for this
treatment protocol.
The aforementioned data showed that the systemic
toxicities of the cyclopropane-containing polyamine
analogues increased with the chain length. The shorter
chain tetramines (7 and 10) were the least toxic, and
their toxicity was comparable to that of the tetramine
SL-11093.13 The longer the chain, as in hexamine 18,
the more the systemic toxicity; the MTD values dropped
from 50 mg/kg to 15 mg/kg when the drug was admin-
istered 5 days in a row (total 250 mg/kg/week for the
tetramines versus 75 mg/kg/week for the hexamine).
When the decamines were assessed, their systemic
toxicity was found to be even higher. For a decamine
with three cyclopropane rings as in 32, the MTD values
dropped to 62.5 mg/kg/week. If only one cyclopropane
was present in the decamine, as in 27, toxicity was even
higher, and the MTD was only 13.2 mg/kg/week. It was
therefore crucial to compare toxicities vs efficacies in
this series of analogues to identify the most effective
therapeutic analogue.
Hexamine 18 was toxic at 25 mg/kg, resulting in two
treatment-related deaths out of three animals. The
deaths occurred after the end of the first treatment
cycle. Dosages of 15 mg and 10 mg/kg were well
tolerated without deaths or body weight loss during the
first treatment cycle. The second treatment cycle, at 15
mg/kg resulted in an average maximum body weight
loss of 8% (2 g). Thus, 15 mg/kg was the MTD for this
treatment protocol.
Efficacy studies were carried out using analogue doses
near the MTD values against established DU-145 tu-
mors (ca. 100-250 mm3) initiating treatment about two
weeks postimplantation. In addition, 7 and 10 were also
studied at doses lower than their MTDs (33.5 mg/kg,
Decamine 32 was lethal at a dosage of 25 mg/kg.
Dosages of 12.5 mg and 6.25 mg/kg were well tolerated
without deaths or body weight loss during the first

Cytotoxic Cyclopropane-Containing Polyamine Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4591
F igu r e 3. Effect of 18, 12.5 mg/kg q1d × 5 in two cycles on
the growth of a DU-145 human prostate tumor xenograft and
body weight in nude mice.
F igu r e 1. Effect of 7, 50 mg/kg, q1d × 5 in two cycles on the
growth of a DU-145 human prostate tumor xenograft and body
weight in nude mice.
and growth remained arrested for at least 10 days after
the end of the second cycle. All experiments utilized
weight mice in each group, including controls. No
xenografted mice were lost when treated with 7, 10, and
18. In the 32 treated group, one mouse died between
days 37 and 41 postimplantation. In the 27 treated
group, one mouse died at the end of first cycle of
treatment and 50% of the mice (4 out of 8) died at the
end of the second treatment cycle. The extent of tumor
growth arrest and body weight loss 10 days after the
end of the second treatment cycle for all the polyamine
analogues are summarized in Figure 6. The mean tumor
volumes in the treated mice as a percent of the mean
tumor volume of controls (vehicle treated mice) at day
41 post-implantation are shown in the upper panel, and
the corresponding mean body weight losses, as com-
pared to the control mice are shown in the lower panel.
While all the analogues are capable of arresting tumor
growth, 10 at the daily 50 mg/kg dose and 18 at the
daily 12.5 mg/kg dose were the two most efficacious
drugs (tumor reductions of ca. 80% were achieved after
two cycles of treatment). Of these two drugs, 10 was
less toxic as judged from the smaller loss of body weight
of animals treated with 10, as compared to that of mice
treated with 18 (Figure 6). Tetramine 10 was, therefore,
chosen for a more detailed assessment of its antitumor
efficacy in vivo.
F igu r e 2. Effect of 10, 50 mg/kg, q1d × 5 in two cycles on
the growth of a DU-145 human prostate tumor xenograft and
body weight in nude mice.
Efficacy studies were carried out using a once-a-
week administration protocol during a four weeks cycle
(q1w × 4). For these studies, 12-14 mice were used to
obtain statistically significant results. The data for
dosages of 50 mg/kg/week and 75 mg/kg/week, along
with their p values determined by using multivariant
analysis,24 are shown in Figure 7. Tetramine 10 was
administered once a week in four- week cycles, inter-
q1d × 5, see Figures 1 and 2, Supporting Information).
The plot of tumor volumes vs time for all five polyamine
analogues are shown in Figures 1-5. With the exception
of 7 at 33.5 mg/kg (Figure 1, Supporting Information),
all compounds showed marked inhibition of tumor
growth within 10 days after the end of the first cycle

4592 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Frydman et al.
F igu r e 4. Effect of 32, 12.5 mg/kg q1d × 5 in two cycles on
the growth of a DU-145 human prostate tumor xenograft and
body weight in nude mice.
F igu r e 6. Summary of effects of polyamine analogues given
q1d × 5 in two cycles on mean tumor volumes and body
weights of nude mice xenografted with DU-145 human pros-
tate tumor cells.
weight. During the same time period, tumor volumes
of mice treated with 75 mg/kg/week were less than 25%
of those in controls. Although an average 20% of body
weight loss was observed at the end of the second cycle
of treatment with this dosage, most of it was regained
within 10 days after the end of the treatment. Thus, in
terms of efficacy, the protocol of prolonged treatment
was as satisfactory as the one involving a more intense
and short-term treatment. Despite the different time
frames, comparable results were obtained with both
regimes of administration; i.e., 75-80% reduction of
tumor volumes with a ca. 20% of body weight loss. In
the case of the short-term administration protocol
(Figure 2), 250 mg/kg of 10 was injected per week, while
in the case of the prolonged treatment (Figure 7) only
75 mg/kg/week was administered.
To assess the efficacy of 10 against large tumors, a
group of seven animals were treated with 75 mg/kg/
week (q1w × 4) 66 days after tumor implantation.
During this period, tumors had grown large, ca. 2000
mm3, as compared to tumor volumes of ca. 200 mm3 that
were the starting points for the data presented above.
Tetramine 10 entirely arrested the growth of the large
tumors (Figure 8). Some body weight loss was observed
for these animals, but the loss was difficult to quantitate
due to the lack of appropriate controls. Mice had to be
sacrificed because of tumor size. Nonetheless, such
marked arrest of the growth of large tumors was
remarkable. After a month of rest and in the absence
of further treatment there was tumor regrowth (Figure
8). The regrowth could be arrested again by a new four-
week cycle of treatment.
F igu r e 5. Effect of 27, 6.25 mg/kg q1d × 5 in two cycles on
the growth of a DU-145 human prostate tumor xenograft and
body weight in nude mice.
rupted by four-week rest periods. After three cycles of
treatment (a total of 120 days after implantation of the
tumors), the weekly dosages of 50 mg/kg and 75 mg/kg
exhibited statistically significant tumor growth inhibi-
tion (p values are between 0.06 and 0.00005). The tumor
volumes in mice treated with 50 mg/kg/week were 70%
of those in the control mice with negligible effect on body
Meta bolism of Tetr a m in e 10. Tumors harvested at
the time intervals shown in Figure 9 after the admin-
istration of 10 already showed significant uptake of the

Cytotoxic Cyclopropane-Containing Polyamine Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4593
F igu r e 7. Growth of DU-145 human prostate tumor xenografts in nude mice and body weights of control untreated mice, mice
treated in three cycles with 10 at a dose of 50 mg/kg q1w × 4, and 75 mg/kg q1w × 4.
compound 6 h after the last ip injection. Maximum drug
uptake occurred 72 h after the last dose and decreased
at 96 h. During the same time frame, two main
metabolites were detected, the monodeethyl derivative
37 and the dideethyl derivative 35 (see Scheme 5 for
structures). Deethylation of 10 is an oxidative dealky-
lation reaction analogous to that observed for SL-
11093.13 The monoethyl derivative 37 was the main
metabolite. In the tumor, 37 peaked 48 h after the last
dose, its levels decreased later, very likely due to
excretion from the tumor. Tetramine 37 was also
detected at high levels in the liver, where rather small
amounts of the parent compound 10 were found 48 h
after the last dosing (Figure 9). In kidney, the main
metabolite was the dideethyl derivative 35 (Figure 9).
Both 37 and 35 are much weaker inhibitors of DU-145
cell growth in culture (their IC50 is ca. 0.60 µM, a 10-
fold decrease of activity as compared to 10).
The effect of 10 on the intracellular pool of the natural
polyamines (putrescine, spermidine, and spermine) in
the tumor suggests it has a long lasting effect on tumor
growth. Six hours after the last ip injection, 10 already
caused a decrease in the polyamine pool present in the
tumor (Figure 10). A burst in the intracellular concen-
trations of the polyamines (particularly spermine) fol-
lowed, that peaked at 24 h after cessation of treatment.
This increase originated very likely from an increased
polyamine uptake from the media. Finally, 96 h after
cessation of treatment, polyamine concentration in the
tumor decreased and stayed low. This delayed strong
inhibitory effect of 10 on polyamine concentration
suggests that the compound has a lasting inhibitory
effect on tumor growth. The intracellular polyamine
pools in the livers of the treated mice were strongly
inhibited by 10. As could be expected from normal
resting cells, the polyamine levels stayed very low after

4594 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Frydman et al.
F igu r e 8. Growth of DU-145 human prostate tumor xenografts in nude mice and body weights of control untreated mice and
mice treated in two cycles with 10 at a dose of 75 mg/kg q1w × 4; treatment started at day 66 after tumor implantation (n )
number of mice).
treatment with 10 (Figure 10). Intracellular polyamine
pools in the kidneys of the treated mice also remained
lower than in control animals after treatment (Figure
10).
restricted rotation (three trans-cyclopropyl residues) was
not as efficacious in inhibiting tumor growth as tetra-
mine 10, where the central butane residue confers
considerable mobility to the structure. Hexamine 18,
although very efficacious in reducing tumor growth, was
too toxic, as measured by the weight loss of the grafted
mice. Decamines 32 and 27 had unfavorable ratios of
efficacy vs toxicity in the grafted mice and were there-
fore not compounds of choice (Figure 6). Of the new
analogues, the RN,ωN-bis(ethyl)tetramine tetrahydro-
chloride 10, had a promising therapeutic index, com-
parable to that of SL-11093. When given ip (dissolved
in saline) to grafted mice that carried established
human prostate cancer tumors at 250 mg/kg/week every
other week for two weeks, the inhibition of tumor
growth (as compared to untreated mice) and mice
weight loss was comparable to that of SL-11093 (Figure
6 and ref 13). There were some differences among both
analogues; SL-11093 intratumoral levels13 were higher
than those of 10 (Figure 9). SL-11093 was also less
N-deethylated to its metabolites in the tumor than was
Con clu sion s
Five new cyclopropane-containing polyamine ana-
logues, 7, 10, 18, 27, and 32, have been assessed for
their activity against DU-145 human prostate cancer
tumors implanted in nude mice. Their structures were
crafted to explore the therapeutic possibilities of
polyamine analogues built around one or more cyclo-
propane rings. The model compound was considered to
be SL-11093, an RN,ωN-bis(ethyl)homospermine ana-
logue where the central butane segment had been
replaced by a trans-1,2-dimethyl cyclopropyl ring.13 SL-
11093 was found to be an efficacious chemotherapeutic
agent against human prostate cancer grown in nude
mice. The new analogues were also, in general, effective
in inhibiting human prostate cancer tumors grafted in
nude mice. Tetramine 7, a polyamine with a highly

Cytotoxic Cyclopropane-Containing Polyamine Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4595
F igu r e 9. Levels of 10 and its metabolites 37 and 35 in the
tumor xenografts, livers, and kidneys of nude mice 96 h after
treatment with a single 50 mg/kg ip dose of 10.
F igu r e 10. Levels of cellular polyamine putrescine, spermi-
dine, and spermine in the tumor xenografts, livers and kidneys
of nude mice 96 h after treatment with a single 50 mg/kg ip
dose of 10.
10. In general, however, both 10 and SL-11093 could
be considered as having similar chemotherapeutic ef-
ficacies.
tively. These metabolites (but not 10) were present in
high concentrations in liver and kidney, suggesting a
catabolic pathway for 10 that leads to its excretion by
dealkylation to more polar metabolites. In conclusion,
tetramine 10 is an alternative choice to SL-11093 in the
selection of potentially useful anticancer polyamine
analogues of reduced toxicity and good efficacy.
Tetramine 10 was also given to grafted mice at a
regime of 75 mg/kg/week during monthly cycles, every
other month for six months. This prolonged treatment
also achieved inhibition of tumor growth of 80% as
compared to untreated mice, and no animal was lost
during or after the treatments. It showed that 10 can
arrest tumor growth (even of large tumors) for long
periods, when administered with the correct protocol.
There was some weight loss (15%) in the treated mice
during this regime, but the mice recovered most of the
lost weight during the monthly resting periods, sug-
gesting the absence of irreversible damage due to
treatment.
The inhibition of tumor growth by 10 can be explained
in part by its accumulation in tumor; as is the case with
SL-11093.13 In liver and kidney, concentrations of 10
remained relatively low, a fact that may account for its
relatively low systemic toxicity. It was deethylated to
its mono- and dideethyl- derivatives 37 and 35, respec-
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
mode; mass spectra (FAB) were obtained on an Autospec (VG)
spectrometer. HPLC analyses of the dansyl derivatives of the
tetramines were routinely performed to check the purity of

4596 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Frydman et al.
the samples. A Vydac C-18 (300 µm pore) column was used
for separations, and a fluorescence spectrometer (340-nm
excitation, 515-nm emission) was used for detection.
added to a stirred mixture of diester 59 (676 mg, 1.45 mmol)
and diamide 4 (1.46 g, 2.95 mmol) in dry DMF (12 mL) at
0 °C. The cooling bath was removed, and stirring was
continued for 10 h. The reaction mixture was quenched with
water (1 mL), acidified to pH 6 with aq HCl (3%), and
concentrated to dryness in vacuo at 40 °C. The residue was
dissolved in ethyl acetate, washed three times with water and
then with brine, and dried (Na2SO4). Column chromatography
(silica gel, hexane:ethyl acetate, 3:1) of the residue left after
evaporation of the organic solution gave tetrasulfonamide 6;
1.26 g (83%). 1H NMR (CDCl3): 0.25-0.50 (m, 6H), 0.70-0.95
(m, 6H), 0.95-1.10 (m, 6H), 2.29 (s, 12H), 2.55 (s, 24H), 2.85-
3.10 (m, 6H), 3.10-3.40 (m, 10H), 6.92 (s, 8H). 13C NMR
(CDCl3): 10.92, 11.15, 12.64, 16.00, 16.16, 16.32, 20.92, 22.67,
40.26, 48.41, 49.04, 49.22, 131.88, 133.23, 133.36, 140.16,
142.21, 142.32. MS-MALDI (m/z) 1073.6 (M+ + Na).
Ter [(E)-5,6,(E)-10,11,(E)-15,16-cyclop r op yl]-3,8,13,18-
tetr a a za eicosa n e Tetr a h yd r och lor id e (7). Tetrasulfona-
mide 6 (1.26 g, 1.2 mmol) was stirred for 10 h in a mixture of
phenol (4.5 g, 48 mmol), HBr (33% in AcOH, 26 mL), and CH2-
Cl2 (13 mL). The mixture was cooled in an ice bath, quenched
with water (5.5 mL), washed two times with CH2Cl2, and
concentrated in vacuo. The residue was neutralized with 2 N
NaOH (1 mL) at 0 °C, made alkaline (pH 12) with 50% KOH
(1 mL), extracted with CHCl3 (five times), dried (Na2SO4), and
concentrated in vacuo. The residue was dissolved in Et2O and
the hydrochloride precipitated with HCl gas. The precipitate
was filtered, washed with Et2O, and dried in vacuo; 374 mg
(66%) of 7 were obtained; mp: 200 °C (dec). 1H NMR (D2O):
δ 0.80-0.92 (m, 6H), 1.15-1.85 (m, 6H), 1.29 (t, J ) 7.3, 6H),
6.28-3.00 (m, 6H), 3.12 (q, J ) 7.3, 4H), 3.10-3.35 (m, 6H).
13C NMR (D2O): δ10.03, 10.14, 10.60, 13.78, 42.69, 50.13,
50.45. MS-ESI (m/z) 323.2 (M+ + 1).
Bis[(E)-5,6-(E)-15,16-cyclop r op yl]-3,8,13,18-t et r a a za -
eicosa n e tetr a h yd r och lor id e (10) was prepared following
the procedures described above for the synthesis of 7. Tetra-
sulfonamide 9 was prepared in 93% yield by condensation of
diester 89 and amide 4. 1H NMR (CDCl3): δ 0.25-0.45 (m, 4H),
0.65-0.85 (m, 4H), 0.99 (t, J ) 7.1, 6H), 1.30-1.42 (m, 4H),
2.29 (s, 12 H), 2.56 (s, 24 H), 2.55-2.95 (m, 4H), 3.05-3.35
(m, 12H), 6.92 (s, 8H). 13C NMR (CDCl3): δ 10.88, 12.65, 15.99,
16.22, 20.91, 22.67, 22.71, 24.42, 40.30, 45.24, 48.41, 48.78,
131.90, 133.33, 140.10, 142.23, 142.30. MS-MALDI 1061.5
(M+ + Na). Treatment of 9 with HBr/AcOH in the presence of
phenol as described for 6 gave 10 (71%); mp: 200 °C (dec); 1H
NMR (D2O) δ 0.85 (t, J ) 7.1, 4H), 1.15-1.30 (m, 4H), 1.26
(t, J ) 7.4, 6H), 1.75-1.90 (m, 4H), 2.82-3.00 (m, 4H), 3.05-
3.30 (m, 12H). 13C NMR (D2O) δ: 10.00, 10.60, 13.75, 22.89,
42.68, 46.65, 50.12, 50.74. MS-ESI (m/z) 311.4 (M+ + 1).
tr a n s-1,2-Bis(br om om eth yl)cyclop r op a n e (13). trans-
1,2- Bis(hydroxymethyl)cyclopropane 128 (2.6 g, 25 mmol) was
dissolved in 60 mL of dry CH2Cl2 under argon, P(Ph)3 (13 g,
50 mmol) was added, and the solution was cooled to 5 °C.
N-Bromosuccinimide (9 g, 50 mmol) was added slowly with
stirring. The mixture was kept at 22 °C for 18 h, the solution
was evaporated to dryness, the residue was extracted with
petroleum ether (bp 35-60 °C) (4 × 25 mL), the pooled extracts
were cooled to 5°C, and the precipitated triphenylphosphine
oxide was filtered off. The solution was evaporated to dryness,
and the residual 13 (4.7 g) was directly used in the next step
without further purification; 1H NMR (CDCl3) δ: 0.85 (t, J )
6.76 Hz, 2H), 1.33 (t, J ) 6.30 Hz, 2H), 3.35 (m, 4H); 13C NMR
(CDCl3) δ: 17.27, 24.41, 36.75.
tr a n s-1-Cya n o-2-(m esitylen esu lfon yloxym eth yl)cyclo-
p r op a n e (2). A solution of ethyl (E)-2-cyanocyclopropanecar-
boxylate 125 (8 g, 57.5 mmol) in isopropyl alcohol (8 mL) was
added over 5 min into an ice-cold, N2 swept solution of LiBH4
(1.254 g, 57.5 mmol) in isopropyl alcohol (40 mL). The reaction
mixture was stirred for 0.5 h in an ice bath. The bath was
then removed, and the reaction was continued at 22 °C for
another 8 h. The reaction mixture was concentrated in vacuo
and evaporated three times from toluene. Et2O (130 mL) and
H2O (2.5 mL) were added, and the reaction mixture was stirred
for 10 h at 22 °C. It was dried (Na2SO4), filtered, and concen-
trated on a rotavapor. The residue of crude 2-cyano-1-hydroxy-
methylcyclopropane was dissolved in pyridine (50 mL) and
cooled to 0 °C, and mesitylene sulfonyl chloride (18.9 g, 86.3
mmol) in pyridine (60 mL) was slowly added into the reaction
mixture. Following 3 h of stirring at 0 °C, the reaction mixture
was poured on ice (500 g) and acidified with 10% HCl, and
the product was extracted with ethyl acetate. The organic
extracts were washed with 3% HCl, a saturated NaHCO3
solution, and brine. Column chromatography (silica gel, hex-
ane:ethyl acetate, 4:1) of the residue left after evaporation of
1
the organic extracts gave 5.25 g (33%) of 2; H NMR (CDCl3)
δ 0.8-1.1 (m, 1H), 1.25-1.40 (m, 2H), 1.70-1.90 (m, 1H), 2.33
(s, 3H), 2.63 (s, 6H), 3.80 (dd, J 1 ) 11.2 Hz, J 2 ) 7.2 Hz, 1H),
4.03 (dd, J 1 ) 11.2 Hz, J 2 ) 5.9 Hz, 1H, 1H), 7.00 (s, 2H); 13
C
NMR (CDCl3) δ 2.28, 11.75, 19.35, 21.01, 22.53, 69.26, 119.90,
126.35, 131.88, 139.82, 143.75; MS-ESI (m/z) 280.2 (M+ + 1).
N-(1-Cya n ocyclop r op ylm eth yl)-N-eth ylm esitylen esu l-
fon a m id e (3). A suspension of NaH (60% in mineral oil, 508
mg, 12.7 mmol) in DMF (15 mL) was added to a stirred
solution of 2 (2.3 g, 8.3 mmol) and N-ethylsulfonamide (2.1 g,
9.3 mmol) at 5 °C and the mixture stirred for 1 h. The cooling
bath was removed, and the reaction mixture was stirred for
additional 10 h. After the mixture was cooled in an ice bath,
it was quenched with H2O, neutralized to pH 7 with 2% HCl,
concentrated in vacuo, suspended in CHCl3, and washed twice
with H2O and brine. Column purification (silica gel, hexane:
ethyl acetate, 4:1) of the residue left on evaporation of the
CHCl3 gave 2.42 g (95%) of the product 3; mp 149-150 °C
(ethyl acetate/hexane); 1H NMR (CDCl3) δ 0.85-1.00 (m, 1H),
1.1 (t, 3H), 1.20-1.30 (m, 1H), 1.60-1.75 (m, 1H), 2.31 (s, 3H),
2.59 (s, 6H), 3.06-3.26 (m, 2H), 3.25-3.34 (m, 2H), 7.00 (s,
2H); 13C NMR (CDCl3) δ 2.75, 12.76, 12.98, 19.66, 20.88, 22.62,
41.06, 47.43, 120.62, 132.03, 132.63, 140.21, 142.76; MS-ESI
(m/z) 307.4 (M+ + 1).
N-E t h yl N,N′-Bis(m esit ylen esu lfon yl)-tr a n s-1,2-b is-
(a m in om eth yl)cyclop r op a n e (4). A solution of nitrile 3 (2.42
g, 7.9 mmol) in Et2O (15 mL) was slowly added into a stirred
suspension of LiAlH4 (368 mg, 9.7 mmol) in Et2O (10 mL) at
0 °C. The mixture was stirred for 2.5 h, the cooling bath was
removed, and the reaction mixture was left stirring for 20 h
at 22 °C. The reaction mixture was quenched with 2 N NaOH
at 0 °C, and the inorganic precipitate was filtered and washed
thoroughly with Et2O. The Et2O solution was dried (Na2SO4)
and concentrated in vacuo. The residue was dissolved in CH2-
Cl2 (30 mL), mesitylenesulfonyl chloride (1.68 g, 7.7 mmol) was
added, and the reaction mixture was cooled on an ice-bath and
treated with 2 N NaOH (16 mL). After 4 h the reaction mixture
was quenched with H2O (60 mL), acidified to pH 1, the product
was extracted with CHCl3, washed with brine, dried (Na2SO4),
and purified on a silica gel column (hexane:ethyl acetate, 4:1);
1.69 g (43%) of 4 were obtained; 1H NMR (CDCl3) δ 0.40-0.52
(m, 2H), 0.94 (t, 3H), 0.95-1.15 (m, 2H), 2.29 (s, 3H), 2.30 (s,
3H), 2.40-2.52 (m, 1H), 2.57 (s, 6H) 2.59 (s, 6H), 2.90-3.45
(m, 5H), 5.58 (t, 1H), 6.94 (4H); 13C NMR (CDCl3) δ 10.10,
12.33, 16.45, 17.51, 20.87, 20.93, 22.55, 22.93, 40.01, 46.61,
47.71, 131.81, 131.88, 132.49, 133.96, 139.08, 140.39, 141.75,
142.52. MS-ESI (m/z) 493.4 (M+ + 1).
Bis(m esitylen esu lfon yl)-12-br om o-10,11-[(E)-1,2-cyclo-
p r op yl]-3,8-d ia za d od eca n e (15). Amide 149 (8.5 g, 18 mmol)
was dissolved in dry DMF (150 mL), and NaH (60% in oil,
0.85 g) was added. The mixture was stirred for 30 min at
22 °C, when 13 (4.0 g, 18 mmol) dissolved in 40 mL of dry
DMF was added and the mixture was further stirred during
18 h. Water (10 mL) was added, the solution was evaporated
to dryness in vacuo, the residue partitioned between CHCl3
and a saturated NH4Cl solution, the organic layer separated,
evaporated to dryness, and the residual oil was purifed by
3,8,13,18-Tetr a k is(m esitylen esu lfon yl)-ter [(E)-5,6,(E)-
10,11,(E)-15,16-cyclopr opyl]-3,8,13,18-tetr aazaeicosan e (6).
Sodium hydride (60% suspension in oil, 161 mg, 4.0 mmol) was

Cytotoxic Cyclopropane-Containing Polyamine Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4597
column chromatography on silica gel using hexane/ethyl
acetate, 1:1 as eluant; 2.7 g (26% yield) of 15 were recovered;
1H NMR (CDCl3) δ: 0.55 (m, 2H), 0.92(m,2H), 1.00 (t, J ) 7.05
Hz, 3H), 1.40 (m, 4H), 2.30 (s, 6H), 2.58 (s, 12H), 2.90-3.20
(m, 10H), 6.94 (s, 4H); 13C NMR (CDCl3) δ: 12.75, 13.74, 19.77,
20.92, 21.07, 22.75, 24.74, 24.80, 37.59, 40.09, 44.62, 45.27,
48.61, 131.91, 140.13, 142.21, 142.43.
of 22 was obtained; 1H NMR (CDCl3) δ: 0.94 (t, 3H), 1.35 (m,
12H), 2.30 (s, 12H), 2.50 (s, 18H), 2.55 (s, 6H), 2.75 (m, 2H),
3.08 (m, 12H), 4.60 (br, 1H), 6.90 (s, 8H).
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 h en eicosa n ecya n id e (23). It was obtained in 98% yield
from 22 following the procedure described for 20. 1H NMR
(CDCl3) δ: 0.95 (t, 3H), 1.39 (m, 12H), 1.83 (m, 2H), 2.20
(t, 2H), 2.30 (s, 12H), 2.59 (s, 24H), 3.10 (m, 16H), 6.98 (s, 8H);
13C NMR (CDCl3) δ: 12.58, 14.47, 20.82, 22.62, 22.69, 23.60,
24.34, 24.42, 24.66, 39.92, 44.46, 44.84, 45.55, 118.64, 131.81,
131.89, 132.04, 133.23, 139.85, 139.90, 142.16, 142.26, 142.69.
3,8,13,18,23,28-Hexa k is(m esitylen esu lfon yl)-10,11-[(E)-
1,2-cyclop r op yl]-20,21-[(E)-1,2-cyclop r op yl]-3,8,13,18,23,-
28-h exa a za tr ia con ta n e (17). Amide 1626 (0.85 g, 1.8 mmol)
was dissolved in 50 mL of dry DMF, NaH (60% in oil, 0.15 g)
was added, the mixture was stirred for 30 min at 22 °C, and
the bromomethyl derivative 15 (2.3 g, 3.7 mmol) dissolved in
25 mL of dry DMF was added. The mixture was stirred for
18 h at 22 °C, and the workup described for 15 was then
followed. Column chromatography on silica gel using chloro-
form:ethyl acetate, 9:1 as eluant gave 2.0 g (34% yield) of 17;
1H NMR (CDCl3) δ: 0.31 (m, 4H), 0.72 (m, 4H), 0.97 (t, J )
7.12 Hz, 6H), 1.26 (m, 12H), 2.29 (s, 18H), 2.54-2.55 (s, 36H),
2.80 (m, 4H), 3.15 (m, 20H), 6.92 (s, 12H); MS-MALDI (m/z);
1567.39 (M+ + Na).
Bis[(10,11-(E)-20,21-(E)-1,2-cyclop r op yl)]-3,8,13,18,23,-
28-h exa a za tr ia con ta n e Hexa h yd r och lor id e (18). Hexa-
mide 17 (2.0 g) was deprotected with hydrogen bromide in
glacial acetic acid following the procedure described for 6. SL-
11231 hexachloride 18 was crystallized from aqueous ethanol;
0.5 g (60% yield) was obtained; 1H NMR (D2O) δ: 0.86 (t, J )
6.8 Hz, 4H), 1.23 (m, 4H), 1.29 (t, J ) 7.3 Hz, 6H), 1.78 (m,
12H), 2.89 (m, 4H), 3.15 (m, 20H); 13C NMR (D2O) δ: 12.48,
12.95, 16.14, 25.28, 45.36, 48.71, 49.05, 49.35, 53.12; MS-ESI
(m/z): 453.6 (M+ + 1).
5,10,15,20-Tetr a k is(m esitylen esu lfon yl)-5,10,15,20-tet-
r a a za -1-d ocosa m in e (24). It was obtained in 98% yield from
1
23 following the procedure described for 21. H NMR (CDCl3)
δ: 0.98 (t, 3H), 1.32 (m, 14H), 1.40 (m,2H), 2.30 (s,12H), 2.55
(s, 24H), 2.60 (m, 2h), 3.05 (m, 16H), 6.95 (s,8H); 13C NMR
(CDCl3) δ: 12.66, 20.87, 22.66, 22.73, 24.49, 24.58, 24.72, 29.58,
40.00, 41.11, 44.52, 44.95, 45.26, 131.92, 133.29, 139.92,
139.97, 142.20, 142.29.
1,5,10,15,20-P en ta k is(m esitylen esu lfon yl)-1,5,10,15,20-
p en ta a za tr icosa n e (25). It was obtained from 24 in 71% yield
1
following the procedure described for 22. H NMR (CDCl3) δ:
0.95 (t, 3H), 1.32 (m, 12H), 2.29 (s, 12H), 2.54 (s, 18H), 2.59
(s, 6H), 2.75 (m, 2H), 3.08 (m, 12H), 4.55 (br, 1H), 6.92 (s, 8H);
13C NMR (CDCl3) δ: 12.61, 20.82, 22.63, 22.71, 22.78, 24.35,
24.45, 24.68, 26.57, 39.96, 41.83, 44.47, 45.08, 131.88, 133.19,
133.28, 133.39, 138.86, 139.91, 141.97, 142.18, 142.25, 142.32;
MS (m/z): 1279.0 (M+ + K).
3,8,13,18,23,28,33,38,43,48-Decakis(m esitylen esu lfon yl)-
25,26-[(E)-1,2-cyclopr opyl]-3,8,13,23,28,33,38,43,48-decaaza-
p en ta con ta n e (26). Pentamide 25 (6.5 g, 5.25 mmol) was
dissolved in 75 mL of dry DMF, and NaH (60%, 0.32 g) was
added in several portions with constant stirring at 22 °C. After
3,8,13-Tr is(m esitylen esu lfon yl)-3,8,13-tr ia za h exa d ec-
ylcya n id e (20). Triamide 1910 (44.1 g, 60 mmol) was dissolved
in 700 mL of dry DMF, the solution cooled to 5 °C, and NaH
(85% in oil, 3.25 g, 120 mmol) added portionwise with constant
stirring. After 30 min, 4-bromobutyronitrile (13.34 g, 90.1
mmol) dissolved in 100 mL of dry DMF was added, and the
stirring was kept for 18 h at 22 °C. The reaction was quenched
with 10 mL of H2O, and the mixture was evaporated to dryness
in vacuo, leaving behind an oil that was purified by chroma-
tography on silica gel using hexane/ethyl acetate, 4:1 as eluent;
47 g (97% yield) of 20 was thus obtained; 1H NMR (CDCl3) δ:
0.94 (t, 3H), 1.40 (m, 8H), 1.89 (m, 2H), 2.25 (t, 2H), 2.31 (s,
8
10 min, diester 5 (1.2 g, 2.6 mmol) dissolved in 45 mL of dry
DMF was slowly added to the stirred solution and the latter
kept for 18 h at 22 °C. Water (5 mL) was added, the solution
adjusted to pH 7 with dil HCl, evaporated to dryness, the
residue partitioned between CHCl3 (200 mL) and water
(100 mL), and the organic layer separated, washed with water
(2 × 100 mL) and evaporated to dryness. The decamide 26
was purified by column chromatography on silica gel using
hexane:ethyl acetate, 7:3, as eluant; 3.0 g (23%) of 26 was
1
obtained; H NMR (CDCl3) δ: 0.30 (t, 2H), 0.70 (t, 2H), 0.98
9H), 2.55 (s, 18H), 3.10 (m, 10H), 3.25 (t, 2H), 6.92 (s, 6H); 13
C
(t, J ) 7.0 Hz, 6H), 1.27 (m, 32H), 2.30 (s, 30H), 2.55 (s, 60H),
3.0 (m, 40H), 6.93 (s, 20H); 13C NMR (CDCl3) δ: 11.04, 12.25,
15.94, 20.93, 22.73, 24.39, 24.78, 40.08, 44.63, 45.19, 48.74,
131.94, 133.26, 140.07, 142.32; MS-MALDI (m/z): 2567.0
(M+ + Na).
NMR (CDCl3) δ: 12.62, 14.57, 20.89, 22.70, 22.78, 23.68, 24.47,
24.72, 39.95, 44.49, 44.91, 119.00, 131.96, 132.12, 139.97,
140.00, 142.24, 142.34, 142.78.
5,10,15-Tr is(m esitylen esu lfon yl)-5,10,15-tr ia za -1-h ep -
ta d ecyla m in e (21). Nitrile 20 (47 g) was dissolved in 100 mL
of CHCl3 and 100 mL of ethanol and reduced with hydrogen
over 4.5 g of PtO2 during 5 h at 45 psi. The catalyst was then
filtered over a Celite pad and the solvent evaporated to
dryness. The residue was purified by chromatography on silica
gel using hexane/ethyl acetate, 7:3 as first eluant, followed by
methanol; 46.2 g (94% yield) of 21 were obtained; 1H NMR
(CDCl3) δ: 0.98 (t, 3H), 1.35 (m, 12H), 2.28 (s, 9H), 2.55 (s,
20H), 3.08 (m, 12H), 6.90 (s, 6H); 13C NMR (CDCl3) δ: 12.67,
14.53, 20.86, 22.66, 22.73, 24.50, 24.57, 24.62, 24.71, 30.37,
40.12, 41.41, 44.54, 45.05, 45.32, 131.85, 133.30, 133.34,
133.43, 139.95, 139.99, 142.18, 142.25.
3,8,13,23,28,33,38,48-Decaaza-[(25,26(E)-1,2-cyclopr opyl]-
p en ta con ta n e Deca h yd r och lor id e (27). Decamide 26 (1 g)
was deprotected using hydrogen bromide (33%) in glacial acetic
acid in the presence of phenol as described for 6, and decahy-
drochloride 27 was obtained in 85% yield. 1H NMR (D2O) δ:
0.88 (t, J ) 7.10 Hz 2H), 1.20 (t, J ) 7.10 Hz, 2H), 1.28 (t,
J ) 7.13 Hz, 6H), 1.78 (m, 32H), 3.10 (m, 40H); MS (m/z):
725.8 (M+ + 1)
3,8,13,18-Tet r a k is(m esit ylen esu lfon yl)-10,11-[(E)-cy-
clop r op yl]-3,8,13,18-t et r a a za oct a d eca n e (28). 1,4-Bis-
(mesitylenesulfonyl)putrescine 16 (5.6 g, 12.4 mmol) and the
bromomethyl derivative 15 (3.9 g, 6.2 mmol) were dissolved
in 150 mL of dry DMF, and NaH (60%, 0.92 g) was added in
one portion. The mixture was stirred for 18 h at 22 °C, it was
then quenched with 10 mL of 10% HCl, the solution evaporated
to dryness, the residue dissolved in CHCl3 (100 mL), the latter
washed first with water (2 × 50 mL) and then with brine (50
mL), the organic solvent was evaporated to dryness, and the
residue purified by column chromatography on silica gel using
CHCl3: ethyl acetate, 9:1, as eluant; 2.3 g (37% yield) of 28
were obtained; mp 63 °C; 1H NMR (CDCl3) δ: 0.35 (m, 2H),
0.80 (m, 2H), 0.95 (t, J ) 5.8 Hz, 3H), 1.30-1.49 (m, 8H), 2.30
(s, 12H), 2.56 (s, 18H), 2.59 (s,6H), 2.70-3.30 (m, 14H), 4.66
(t, J ) 6.4 Hz, 1H), 6.93 (s, 8H); MS-MALDI (m/z); 1021.0
(M+ + Na).
1,6,11,16- Tetr akis(m esitylen esu lfon yl)-6,11,16-tetr aaza-
1-h ep ta d ecyla m in e (22). Heptadecylamine 21 (46.2 g, 57.4
mmol) was dissolved in a mixture of 270 mL of CHCl3 and
137 mL of 2 N NaOH, the mixture was cooled to 5 °C, and a
solution of mesitylenesulfonyl chloride (13.2 g, 60 mmol) in
80 mL of CHCl3 was slowly added with stirring. The mixture
was kept for further 18 h at 22 °C, the organic layer was then
separated, the aqueous solution was further extracted with
CHCl3 (2 × 150 mL), and the organic solutions were combined,
washed with H2O (2 × 350 mL) and then with brine (200 mL),
and evaporated to dryness. The residue was purified by column
chromatography on silica gel, using hexane/ethyl acetate, 7:3,
as a first eluant followed by ethyl acetate; 43.0 g (79% yield)

4598 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Frydman et al.
Bis(m esitylen esu lfon yl)-1,4-tr a n s-1,2-d ia m in om eth yl-
cyclop r op a n e (29). Bis(bromomethyl)cyclopropane 13 (1.14
g, 5 mmol) and mesitylenesulfonamide (2.19 g, 11 mmol)
dissolved in 25 mL of anhydrous DMF were cooled to 5 °C,
and NaH (60% in oil, 0.48 g) was added in several portions
under an argon atmosphere. The mixture was stirred at 22°C
during 18 h, after which it was evaporated to dryness in vacuo,
and the residue was partitioned between ethyl acetate (50 mL)
and water (50 mL). The organic layer was separated and
evaporated to dryness, and the residue was purified by column
chomathography on silica gel using ethyl acetate/hexane, 3:7,
as eluant; 1.37 g (47%) of 29 were obtained; mp 152-153,°C
(ethyl acetate/hexane); 1H NMR (CDCl3) δ: 0.06-0.10 (m, 1H),
0.62-0.75 (m, 1H), 1.00-1.16 (m, 2H), 2.29 (s, 6H), 2.62 (s,
12H), 2.69 (dd, J ) 5.0, 8.6 Hz, 2H), 3.04 (dd, J ) 5.0, 13.2
Hz, 2H), 3.70 (br, 2H), 6.04 (s, 4H).
5,10-Bis(m esitylen esu lfon yl)-1,14-d ibr om o-7,8-[(E)-1,2-
cyclop r op yl]-5,10- d ia za tetr a d eca n e (30). Bis(mesitylene-
sulfonyl)-trans-1,2-diaminomethylcyclopropane 29 (1.4 g, 5.2
mmol) was dissolved in dry DMF (50 mL) under nitrogen, and
NaH (60%, 0.4 g) was added to the solution. After the mixture
was stirred for10 min at 22 °C, 1,4-dibromobutane (6.7 g, 31
mmol) dissolved in 25 mL of dry DMF was added in one
portion. After the solution was stirred for further 18h, water
was added (5 mL) and the reaction mixture was evaporated
to dryness. The residue was purified by column chromatog-
raphy on silica gel using hexane:ethyl acetate, 4:1, as eluant;
1.5 g (68% yield) of 30 were obtained; 1H NMR (CDCl3) δ: 0.41
(t, J ) 6.9 Hz, 2H), 0.82 (t, J ) 6.0 Hz, 2H), 1.66 (m, 8H), 2.29
(s, 6H), 2.58 (s, 12H), 2.94 (m, 2H), 3.21 (m, 10H), 6.94 (s, 4H);
13C NMR (CDCl3) δ: 11.04, 16.14, 20.93, 22,74, 25.70, 29.65,
32.88, 44.74, 48.88, 131.97, 160.12, 142.48; MS-MALDI (m/z):
757.09 (M+ + Na).
3,8,13,18,23,28,33,38,43,48-Decakis(m esitylen esu lfon yl)-
10,11-25,26-40,41-ter [(E)-1,2-cyclop r op yl]-3,8,13,23,28,33,-
38,43,48-d eca a za p en ta con ta n e (31). Tetramide 28 (1.4 g,
1.4 mmol) was dissolved in 50 mL of dry DMF, and while kept
under argon, NaH (60%, 75 mg) was added. The mixture was
stirred for 10 min at 22 °C, a solution of the dibromo derivative
30 (0.5 g, 0.7 mmol) in 25 mL of dry DMF was then added,
and the mixture was stirred for 18 h at 22 °C. Water (5 mL)
was added, the solution adjusted to pH 7 with dilute HCl,
evaporated to dryness in vacuo, and the residue purified by
column chromatography on silica gel using chloroform:ethyl
acetate, 9:1 as eluant; 1.0 g (28%) of 31 was obtained; 1H NMR
(CDCl3) δ: 0.29 (m, 6H), 0.70 (m, 6H), 0.96 (t, J ) 6.98 Hz,
6H), 1.26 (m, 24H), 2.28 (s, 30H), 2.53, 2.54, 2.55 (s,s,s, 60H),
2.72 (m, 6H), 3.09 (m, 34H), 6.91(s, 20H); 13C NMR(CDCl3)
δ:11.03,12.73, 15.88, 20.93, 22.72, 24.39, 40.07, 44.61, 44.89,
48.89, 48.72, 131.95, 133.27, 140.04, 142.34; MS-MALDI
(m/z) 2594.1 (M+ + Na).
2H), 1.40-1.65 (m, 6H), 2.32 (s, 6H), 2.58 (s, 12H), 2.96 (dd,
J 1 ) 15.2 Hz, J 2 ) 7.4 Hz, 2H), 3.15 (dd, J 1 ) 15.4 Hz, J 2
)
6.2 Hz, 2H,), 3.18-3.38 (m, 4H), 6.98 (s, 4H); 13C NMR (CDCl3)
δ 2.71, 12.77, 19.35, 20.95, 22.72, 24.41, 45.89, 47.90, 120.54,
132.17, 132.64, 140.16, 142.99; MS-ESI (m/z) 628.8 (MNH4+),
611.6 (MH+).
N,N′-Bis[2-(Mesitylen esu lfon yla m in om eth yl)cyclop r o-
p ylm et h yl]-N,N′-b is(m esit ylen esu lfon yl)-1,4-b u t a n ed i-
a m in e (34). A suspension of LiAlH4 (365 mg, 9.6 mmol) in
dry THF (10 mL) was added to an ice cold solution of dinitrile
33 (2.77 g, 4.54 mmol) in dry THF (20 mL) and the mixture
stirred for 1 h at 0 °C followed by further stirring for 48 h at
22 °C. The reaction mixture was cooled in an ice-bath,
quenched with 2 N NaOH until a white precipitate formed,
and the mixture was diluted with 3 volumes of CH2Cl2. The
suspension was dried (Na2SO4) and filtered through a Celite
cake. The filtrate was concentrated in vacuo and the residue
dissolved in light petroleum ether and evaporated to dryness.
This operation was repeated three times; N,N′-bis[(amino-
methyl)cyclopropylmethyl]-N,N′-bis(mesitylenesulfonyl)-1,4-
butanediamine thus obtained was further utilized without
additional purification. NaOH (2 N) (10 mL) was added at
5 °C to a mixture of the bisamine and mesitylsulfonyl chloride
(2.1 g, 9.5 mmol) in CH2Cl2 (40 mL). The ice bath was removed,
and the reaction was stirred overnight at 22 °C. It was
quenched with aq NH4Cl and diluted three times with CHCl3,
and the product was washed with H2O, NaHCO3, and brine.
Column purification (silica gel,CHCl3:EtOAc, 9:1) gave 34, 1.86
1
g (42%), mp 90 °C (ethyl acetate/hexane). H NMR (CDCl3) δ
0.30-0.50 (m, 4H), 0.85-1.10 (m, 4H), 1.10-1.22 (m, 4H), 2.23
(s, 6H), 2.31 (s, 6H), 2.35-2.51 (m, 2H), 2.53 (s, 12H), 2.62 (s,
12H), 2.80-3.30 (m, 10H), 5.52 (t, J ) 5.27 Hz, 2H,), 6.93 (s,
8H); 13C NMR (CDCl3) δ 10.06, 16.07, 17.62, 20.85, 20.94,
22.63, 22.89, 23.96, 44.88, 46.48, 48.30, 131.82, 131.94, 132.40,
133.95, 139.00, 140.25, 141.79, 142.65; MS-ESI (m/z) 1021.6
(MK+), 1005.6 (MNa+), 1001.0 (MNH4+), 983.6 (MH+).
N′′-Eth yl-N,N′-Bis[2-(Mesitylsu lfon yla m in om eth yl)cy-
clop r op ylm et h yl]-N,N′-b is(m esit ylen esu lfon yl)-1,4-b u -
t a n ed ia m in e (36). Sodium hydride (60% suspension in
mineral oil, 38.4 mg, 0.96 mmol), was added into an ice-cold
stirred mixture of 34 (900 mg, 0.915 mmol) in DMF (18 mL)
and stirred for 10 min. Iodoethane (171.2 mg, 1.1 mmol) was
added into the reaction mixture, the cooling bath was removed,
and the reaction was stirred for 10 h at 22 °C. The reaction
mixture was quenched with 0.5 mL of H2O, concentrated in
vacuo, dissolved in CHCl3, washed with brine, and dried (Na2-
SO4). Column chromatography purification (silica gel, CHCl3:
1
EtOAc, 23:2) gave 36, 413 mg (45%). H NMR (CDCl3) δ 0.2-
0.4 (m, 1H), 0.38 (t, J ) 6.9 Hz, 3H), 0.6-0.8 (m, 2H), 0.8-
0.95 (m, 2H), 0.98 (t, J ) 7.1 Hz, 3H), 1.15-1.40 (m, 4H), 2.29
(s, 12H), 2.30-2.50 (m, 2H), 2.54 (s, 6H), 2.54 (s, 6H), 2.56 (s,
6H), 2.62 (s, 6H), 2.70-3.35 (m, 12H), 5.52 (t, J ) 5.4 Hz, 1H),
6.93 (s, 8H); 13C NMR (CDCl3) δ 10.01, 10.80, 12.58, 15.84,
16.17, 16.23, 17.60, 20.91, 22.65, 22.91, 24.06, 24.25, 40.23,
44.96, 45.08, 46.54, 48.31, 48.65, 131.89, 132.43, 133.25,
139.04, 140.03, 140.09, 140.32, 141.75, 142.27, 142.34, 142.60;
MS-ESI (m/z)1033.6 (MNa+), 1011.6 (MH+).
Ter -[(10,11(E),25-26(E),40,41(E)-(1,2-cyclop r op yl)]-3,8,-
23,28,33,38,43,48-d eca a za p en ta con ta n e Deca h yd r och lo-
r id e (32). Decamide 31(1.0 g) was deprotected with hydrogen
bromide (33%) in glacial acetic acid in the presence of phenol
as described for 6. Decahydrochloride 32 was obtained in 80%
1
yield (0.3 g); H NMR (D2O) δ: 0.85 (t, 6.9 Hz, 6H), 1.23 (t,
J ) 6.9 Hz, 6H), 1.28 (t, J ) 7.22 Hz, 6H), 1.78 (m, 24H), 2.89
(m, 6H), 3.10 (m, 34H); 13C NMR (D2O) δ: 12.65, 13.10, 16.28,
25.43, 45.51, 48.86, 49.21, 49.53, 53.28; MS-ESI (m/z) 750
(M+ + 1).
Bis-[(E)-5,6,(E)-15,16-(1,2-cyclop r op yl)]-3,8,13-h ep t a -
d eca m in e Tetr a h yd r och lor id e (37). HBr in acetic acid
(30%, 9 mL) was added to an ice-cold solution of 36 (410 mg,
0.41 mmol) and phenol (1.5 g, 16.2 mmol) in CH2Cl2 (4.5 mL),
stirred for 15 h at 22 °C, and then cooled to 0 °C and quenched
with 10 mL of H2O. The aqueous phase was washed twice with
CH2Cl2, concentrated in vacuo, cooled on the ice bath, and
made basic with 2 N NaOH (5 mL). The product was extracted
with CHCl3 (six times), dried (Na2SO4), concentrated, dissolved
in EtOH (2 mL), and cooled on an ice bath. HCl (37%, 2 mL)
was added, and the precipitate was collected, washed with
EtOH, and dried in vacuo; 142 mg (82%) of 37 were collected.
1H NMR (D2O) δ 0.84 (m, 4H), 1.12-1.25 (m, 4H), 1.29 (t, J )
7.3 Hz, 3H), 1.70-1.90 (m, 4H), 2.75-2.95 (m, 4H), 3.05-3.30
(m, 10H); 13C NMR (D2O) δ 9.83, 10.00, 10.62, 13.76, 15.02,
22.93, 42.71, 42.87, 46.67, 49.00, 50.15, 50.78; MS-ESI (m/z)
283.4 (MH+).
N,N′-Bis(2-Cya n ocyclop r op ylm eth yl)-N,N′-bis(m esity-
len esu lfon yl)-1,4-bu ta n ed ia m in e (33). NaH (60% suspen-
sion in mineral oil, 640 mg,16 mmol) was added into a solution
of 16 (3 g, 6.63 mmol) in DMF (200 mL) at 0 °C and stirred
for 10 min. Ester 16 (3.9 g, 13.92 mmol) was introduced and
stirred for 2 h, the cooling bath was removed, and the reaction
was left overnight at 22 °C. The reaction was cooled on the
ice bath, quenched with 1 mL of H2O, concentrated to dryness
in vacuo, and dissolved in EtOAc. The organic solution was
washed with H2O, 3% HCl, saturated NaHCO3, and brine,
dried (Na2SO4), and purified by column chromatography (silica
gel, EtOAc:CHCl3,1:9); 2.9 g (72%) of 33 was obtained. 1H NMR
(CDCl3) δ 0.75-0.9 (m, 2H), 0.95-1.10 (m, 2H), 1.15-1.30 (m,

Cytotoxic Cyclopropane-Containing Polyamine Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4599
1,14-Dia m in e-bis-[(E)-2,3,(E)-12,13(1,2-cyclop r op yl)]5,-
10-d ia za tetr a d eca n e tetr a h yd r och lor id e (35) was pre-
pared by deprotection of 34 (400 mg, 0.407 mmol) in CH2Cl2
(4.5 mL) with phenol (1.523 g, 16.2 mmol) and HBr (9 mL,
30% in acetic acid) as described for 37; 147 mg (90%) of 35
in a barrier facility. Either tumor cell suspension (1 × 106) or
tumor fragments (ca. 2 × 3 mm3) were implanted subcutane-
ously with a 25-gauge regular needle. Tumor size was mea-
sured twice per week in two perpendicular dimensions with a
vernier caliper and converted to tumor volume using the
formula: (l × w2)/2, where l and w refer to the longer and
shorter dimensions, respectively. Animal body weights were
measured twice per week at the same time intervals as the
tumor dimension measurements, and mortality was monitored
daily. Effects of treatment on tumor growth, expressed as %
growth delay, were calculated following the relation:
1
were obtained. H NMR (D2O) δ 0.83 (m, 4H), 1.15-1.30 (m,
4H), 1.75-1.90 (m, 4H), 2.75-2.95 (m, 4H), 3.05-3.30 (m, 8H);
13C NMR (D2O) δ 9.84, 13.75, 15.02, 22.94, 42.88, 46.67, 49.01,
50.85; MS-ESI (m/z) 255.6 (MH+).
Cell Cu ltu r e. DuPro cells were obtained from Dr. M. Eileen
Dolan, University of Chicago, Department of Medicine. 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.
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
polyamine analogues. 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 and 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 An a lysis. 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 centrifuging 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 ap-
propriate volume of the supernatant (50-100 µL) was fluo-
rescence-labeled by derivatization with dansyl chloride fol-
lowing procedures published elsewhere.27 Each sample was
loaded onto a C-18 high-performance liquid chromatography
column and separated at the analytical laboratory of the
University of Wisconsin Comprehensive Cancer Center (UWC-
CC) using a previously published procedure.27 Peaks were
detected and quantitated using a Shimadzu HPLC fluores-
cence monitor coupled to a Spectra-Physics peak integrator.
Because polyamine levels vary with environmental conditions,
control cultures were sampled for each experiment.
MTT Assa y. 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 atmo-
sphere. Twenty microliters of appropriately diluted stock
solutions of each drug was added to the middle eight 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.
Twenty five microliters of a 5 mg/mL solution of 3-(4,5-
dimethythiazol-2yl)-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
contains: 100 g lauryl sulfate (SDS), 250 mL of N,N-dimeth-
ylformamide in 2 mL of glacial acetic acid, pH 4.8). The color
that developed was read at room temperature at 570 nm in a
E-max Precision Microplate Reader (Molecular Devices Cor-
poration, Sunnyvale, CA), and data were analyzed using
manufacturer supplied cell survival software.
% growth delay ) [(T - C)/C] × 100%
where T and C represented the median times post-staging for
treated (T) and control (C) tumors to attain a prescribed size
depending on the model. Analogues were administered intra-
peritoneally (ip) in water using either a chronic treatment
schedulesadministration for several cycles of once a day for
five consecutive days (q1d × 5) with a rest period of ca. 10
days between each cycle, or a once a week administration
schedule during monthly cycles with a month rest period in
between. Treatments were usually initiated ca. 15 days after
tumor implantation when the mice had established tumors
ranging in size from 100 to 245 mm3.
Meta bolic Stu d ies. Nude mice implanted with human
prostate Du-145 cancer cell line were kept under the conditions
described in the previous section. When tumor volumes
reached sizes of ca. 200 mm3, they were divided into two groups
of three animals each; group 1 was given ip water for injection
and group 2 was treated ip once a day for 5 days with 50 mg/
kg/day of 10. The animals were bled by retro-orbital puncture
under CO2/O2 anesthesia 6 h after the last injection, and
tumor, liver, and kidneys were harvested at 6, 24, 48, and 96
h after the last dose and snap frozen in liquid nitrogen. The
tissues were homogenized in 1 mL of PBS using a Power Gen-
700 homogenizer and centrifuged at 100 000 rpm for 10 min
at 4 °C. After centrifugation, the pellet was resuspended in
0.5 mL of 2% perchloric acid and stored overnight at -20 °C.
The pellet was sonicated with three 30 s pulses and then
centrifuged at 10 000 rpm for 10 min at 4 °C. The supernatant
was collected, and its polyamine composition was analyzed by
derivatization with dansyl chloride followed by HPLC separa-
tion described above. Each dansyl polyamine derivative was
collected and its molecular weight determined by mass spec-
trometry (MS-ESI).
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 Mr Herbert Grimek
for performing the HPLC analyses. This work was
performed at the Mass Spectrometry-Bioanalytical
Facility of the Biotechnology Center of the University
of Wisconsin.
Su p p or tin g In for m a tion Ava ila ble: Figures showing
effect of 7 and 10 on growth of DU-145 human prostate tumor
xenograft. This material is available free of charge via the
Internet at http://pubs.acs.org.
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