Synthesis and evaluation of hydroxylated polyamine analogues as antiproliferatives

Article abstract of DOI:10.1021/jm000532q

A new means of accessing N¹-cyclopropylmethyl-N¹¹-ethylnorspermine (CPMENSPM) and the first synthesis of (2R,10S)-N¹- cyclopropylmethyl-2,10-dihydroxy-N¹¹-ethylnorspermine [(2R,10S)-(HO)₂CPMENSPM] are

Full text of DOI:10.1021/jm000532q

J . Med. Chem. 2001, 44, 2451-2459
2451
Syn th esis a n d Eva lu a tion of Hyd r oxyla ted P olya m in e An a logu es a s
An tip r olifer a tives
Raymond J . Bergeron,* Ralf Mu¨ller, Guangfei Huang, J ames S. McManis, Samuel E. Algee, Hua Yao,
William R. Weimar, and J an Wiegand
Department of Medicinal Chemistry, J . Hillis Miller Health Science Center, University of Florida, Gainesville, Florida 32610
Received November 22, 2000
A new means of accessing N1-cyclopropylmethyl-N11-ethylnorspermine (CPMENSPM) and
the first synthesis of (2R,10S)-N1-cyclopropylmethyl-2,10-dihydroxy-N11-ethylnorspermine
[(2R,10S)-(HO)2CPMENSPM] are described. Both of these polyamine analogues are shown to
be more active against L1210 murine leukemia cell growth than either N1,N11-diethylnorsper-
mine (DENSPM) or (2R,10R)-N1,N11-diethyl-2,10-dihydroxynorspermine [(2R,10R)-(HO)2DENSPM]
after 96 h of treatment; the activity was comparable to that of (2S,10S)-N1,N11-diethyl-2,10-
dihydroxynorspermine [(2S,10S)-(HO)2DENSPM] at 96 h. Both cyclopropyl compounds reduced
putrescine and spermidine pools, but less effectively than did DENSPM and its derivatives.
Only CPMENSPM, and not (2R,10S)-(HO)2CPMENSPM, lowered spermine pools. As with
DENSPM and (2R,10R)-(HO)2DENSPM, both cyclopropyl analogues diminished ornithine
decarboxylase and S-adenosylmethionine decarboxylase activity. Unlike the hydroxylated
DENSPM compounds, both cyclopropyl norspermines substantially upregulated spermidine/
spermine N1-acetyltransferase. The most interesting effect of hydroxylating CPMENSPM is
the profound reduction in toxicity compared with that of the parent drug. The same phenomenon
had been observed for the DENSPM/(2R,10R)-(HO)2DENSPM pair. Thus, hydroxylation of
norspermine analogues appears to be a way to maintain the compounds’ antiproliferative
activity while reducing their toxicity.
In tr od u ction
related to their ability to stimulate SSAT.17-19 The
tetraamine DENSPM upregulates SSAT by 15-fold in
L1210 murine leukemia cells, yet DESPM and DEH-
SPM stimulate SSAT by 4.6- and 1.4-fold, respectively.17
The SSAT stimulation is even more striking in other
cell lines.23 Furthermore, although the Ki values of
DESPM and DEHSPM for the polyamine transport
system are within error of each other, the DENSPM
value is >10 times as great.21
Minor changes in an analogue’s structure also cause
a significant difference in pharmacological behavior in
whole animals. DENSPM has little gastrointestinal
antitransit activity; DEHSPM is a potent antidiar-
rheal.24 DEHSPM also decreases blood pressure in rats,
whereas neither DENSPM25 nor DESPM possesses this
property. All three analogues are first metabolized in
vivo by N-deethylation to the corresponding tetra-
amine.26-29 Spermine and norspermine are then pro-
cessed by deaminopropylation26,28,29 through the poly-
amine metabolic network. However, because of the
presence of the aminobutyl fragments in homosper-
mine,27 it is not terminally N-acylated and cannot be
further degraded. Thus, it remains unmetabolized for
a protracted period of time in animals. These differences
in susceptibility to catabolism correlate with the toxicity
profiles: DENSPM is the least toxic,26 and DEHSPM
is the most.27
The observed increases in polyamine biosynthesis in
neoplastic tissues have made the polyamine biosynthetic
network an attractive target in cancer chemotherapeutic
strategies.1-3 There is abundant evidence that N-alkyl-
ated polyamines exhibit activity against a number of
murine and human tumor lines both in vitro and in
vivo.4-8 The phase II clinical trials with N1,N11-dieth-
ylnorspermine (DENSPM) have further highlighted
interest in these molecules. It is certainly well estab-
lished that these polyamine analogues utilize the poly-
amine transport apparatus for incorporation into cells,9-11
reduce polyamine pools,12 diminish the level of ornithine
decarboxylase (ODC)13,14 and S-adenosylmethionine de-
carboxylase (AdoMetDC) activities,14,15 and in some
cases upregulate spermidine/spermine N1-acetyltrans-
ferase (SSAT).16-19 However, the precise mechanism by
which the molecules induce death in tumor cells has not
been completely defined.20 Systematic structure-
activity studies have shown that very small structural
alterations in these compounds can cause pronounced
differences in their biological properties at both cellular
and whole animal levels.12,21,22 On a cellular level, for
example, whereas the tetraamines DENSPM, N1,N12
-
diethylspermine (DESPM), and N1,N14-diethylhomo-
spermine (DEHSPM) suppress ODC and AdoMetDC to
about the same level at equimolar concentrations, the
impact of both DESPM and DEHSPM on cell growth is
much faster than that observed for DENSPM. The most
profound difference among the three analogues is
Although DENSPM’s chronic dose-limiting toxicity is
gastrointestinal, its subacute toxicity is neurological.
Considering the enormous difference between the dose
given to patients and the dose at which mice exhibited
neurological side effects, including pronounced ataxia,
* Author to whom correspondence should be addressed [telephone
(352) 846-1956; fax (352) 392-8406; e-mail bergeron@mc.cop.ufl.edu].
10.1021/jm000532q CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/21/2001

2452 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Bergeron et al.
Sch em e 1. Synthesis of Diamine Reagents (5a -c)a
F igu r e 1. Structures of N1-cyclopropylmethyl-N11-ethylnor-
spermine (CPMENSPM, 1) and its hydroxylated derivative,
(2R,10S)-N1-cyclopropylmethyl-2,10-dihydroxy-N11-ethylnor-
spermine [(2R,10S)-(HO)2CPMENSPM, 2].
intention tremors, and motor dysfunction,30 it seemed
that neurotoxicity would be an unlikely problem in the
clinic. Nevertheless, this represented an easily observ-
able means to help evaluate how structural alterations
in the polyamine backbone affect toxicity. In a recent
study,30 we examined the effect of hydroxylation of
DENSPM on both the activity of the compound against
L1210 cells in culture and adverse effects in vivo. In
this paper, we have carried out these observations
further to include an unsymmetrically substituted
analogue, N1-cyclopropylmethyl-N11-ethylnorspermine
[CPMENSPM (CPENSpm in the nomenclature of Woster
and co-workers),31 1; Figure 1]. This tetraamine is
cytotoxic to several tumor cell lines in vitro, increasing
SSAT activity31-33 and initiating apoptosis.31,32,34-36
However, its activity and toxicity in vivo have not been
reported. We describe an improved method of generating
CPMENSPM and the synthesis of a hydroxylated
derivative, (2R,10S)-N1-cyclopropylmethyl-2,10-dihydroxy-
N11-ethylnorspermine [(2R,10S)-(HO)2CPMENSPM, 2;
Figure 1]. Initial evaluations of these analogues in
L1210 cells are performed; the toxicity of these com-
pounds in mice is also examined. Once again, hydroxy-
lation is shown to substantially reduce toxicity.
a
Reagents: (a) mesitylenesulfonyl chloride, NaOH (aq), CH2Cl2,
88%; (b) NaH, DMF, X(CH2)3X, X ) Cl (66%), X ) Br (71%), X )
I (40%), X ) OTs (0%).
Sch em e 2. Synthesis of CPMENSPM (1)a
Resu lts
Syn th esis. The first synthesis of the polyamine
analogue CPMENSPM (1) was carried out in 2% overall
yield.31 Specifically, N-benzyl-1,3-diaminopropane37 was
converted into 21 mg of 1 as its tetrahydrobromide salt
in 10 steps.31 To generate sufficient quantities of
CPMENSPM for biological testing in our laboratories,
dialkylated polyamine 1 as its tetrahydrochloride salt
was accessed by a segmented route (Schemes 1 and 2).
N-Ethyl-1,3-diaminopropane (3) was treated with mes-
itylenesulfonyl chloride (2 equiv) under biphasic condi-
tions (CH2Cl2/dilute NaOH), giving 4 in 88% recrystal-
lized yield (Scheme 1). Dropwise addition of the anion
of 4 (NaH/DMF) to 1,3-dichloro-, 1,3-dibromo-, or 1,3-
diiodopropane in 12-fold excess in DMF gave each
electrophilic reagent (5a , 5b, and 5c) in 66, 71, and 40%
yield, respectively. However, addition of deprotonated
4 to excess 1,3-propanediol-di-p-tosylate led to a mixture
from which tosylate 5d could not be easily purified.
Synthesis of the segment that contains the small ring
began with modification of (aminomethyl)cyclopropane
hydrochloride (6) using mesitylenesulfonyl chloride
(CH2Cl2/1 N NaOH) in 97% yield (Scheme 2). Proton
removal from the resulting 7 with NaH in DMF and
alkylation with N-(3-bromopropyl)phthalimide in DMF
provided intermediate 8 in 84% yield. Unmasking the
primary amino functionality of 8 with hydrazine in
a
Reagents: (a) mesitylenesulfonyl chloride, NaOH (aq), CH2Cl2,
97%; (b) N-(3-bromopropyl)phthalimide, NaH, DMF, 84%; (c)
hydrazine hydrate, EtOH, reflux, 87%; (d) a, 84%; (e) 5b, NaH,
DMF, 73%; (f) 30% HBr in HOAc, PhOH, CH2Cl2; NaOH; HCl,
36%.
refluxing ethanol generated primary amine 9 in 87%
yield. Treatment of 9 with mesitylenesulfonyl chloride
as before produced N,N′-bis(mesitylenesulfonyl)-N-cy-
clopropylmethyl-1,3-diaminopropane (10) in 84% yield.
Connecting disulfonamide 10, which contains the
cyclopropylmethylamino unit, with halides 5a -c, which

Hydroxylated Polyamine Analogues as Antiproliferatives
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2453
Sch em e 3. Synthesis of (2R,10S)-(HO)2CPMENSPM
(2)a
possess the ethylamino functionality, would complete
the framework of unsymmetrically dialkylated norsper-
mine 1. A solution of the anion of 10 (NaH/DMF) was
added to a solution of each alkylating agents5a , 5b, or
5csin DMF to optimize production of masked tet-
raamine 11. With alkyl chloride 5a , difficulties in
purification prevented isolation of 11. Utilizing iodo
compound 5c resulted in 11 in only 6% yield. Treatment
of 10 with primary bromide 5b led efficiently to tet-
ramesitylenesulfonamide 11 (73% yield). Removal of the
protective groups from 11 using 30% HBr in acetic acid
and phenol in CH2Cl2 and conversion to the tetrahy-
drochloride salt furnished CPMENSPM (1) in 36%
recrystallized yield.
We report the first synthesis of (2R,10S)-(HO)2-
CPMENSPM (2) in which both outer trimethylene
chains of CPMENSPM (1) are hydroxylated. Two pairs
of enantiomeric dihydroxylated, terminally diethyl-
ated tetraamines have been synthesized by way of frag-
ments in this laboratory: (2R,10R)- and (2S,10S)-N1,N11-
diethyl-2,10-dihydroxynorspermine [(HO)2DENSPMs]
and (3R,12R)- and (3S,12S)-N1,N14-diethyl-3,12-dihy-
droxyhomospermine.30 The final steps in the assembly
of (2S,10S)-(HO)2DENSPM involved alkylation of N,N′-
dibenzyl-1,3-propanediamine with N-[(2S)-2,3-epoxypro-
pyl]-N-ethyltrifluoromethanesulfonamide (2 equiv) and
removal of the trifluoromethanesulfonyl (Tf)38 and ben-
zyl (Bn) protecting groups with LiAlH4 (THF, reflux, 19
h) and H2 (1 atm, 10% Pd-C, 1 N HCl, EtOH), respec-
tively.30 However, because a cyclopropyl group may
undergo ring opening under reductive conditions,39-41
it was not initially clear that we could use this meth-
odology to make (2R,10S)-(HO)2CPMENSPM (2). The
stability of the cyclopropane ring to these deprotection
conditions was verified in two model systems before the
synthesis of hydroxylated tetraamine 2 was undertaken.
First, N-benzyl-N-cyclopropylmethyl-N-propylamine was
prepared by successive alkylation of primary amine 6
with benzaldehyde (NaBH4/EtOH)42 and propyl iodide
(K2CO3/1-butanol). Mild hydrogenolysis of this tertiary
amine (1 atm, 10% Pd-C, 1 N HCl, EtOH) gave N-cyclo-
propylmethyl-N-propylamine in 91% yield. Second, heat-
ing the triflamide of N-cyclopropylmethyl-N-propyla-
mine with LiAlH4 (THF, reflux, 3 days) or sodium bis(2-
methoxyethoxy)aluminum hydride (Red-Al) in boiling
toluene for 12 h43 removed the sulfonyl group to give
back the secondary amine in 60 or 75% yield, respec-
tively. These model reactions demonstrated that the
three-membered ring would be stable to the reductive
amine deprotection steps during the synthesis of 2.
a
Reagents: (a) Tf2O, Et3N, CH2Cl2, 50%; (b) (S)-(+)-2,2-
dimethyl-1,3-dioxolane-4-methanol, PPh3, diisopropyl azodicar-
boxylate, THF, 96%; (c) 1 N HCl, acetone, reflux, 98%; (d) TsCl,
CH2Cl2, pyridine, 88%; (e) K2CO3, CH3OH, 74%; (f) N,N′-dibenzyl-
1,3-diaminopropane, EtOH, reflux, 64%; (g) EtOH, reflux, 94%;
(h) Red-Al, toluene, reflux, 60%; (h) H2, 10% Pd-C, 1 N HCl (8
equiv), EtOH, 70%.
of 13 in refluxing 1 N HCl (acetone) gave diol 14 in 98%
yield; activation of the primary hydroxyl group of 14
with tosyl chloride in pyridine led to monotosylate 15
in 88% yield. Intramolecular displacement of the tosy-
late by the secondary alcohol of 15 under basic condi-
tions (K2CO3/CH3OH) at room temperature furnished
epoxide 16 in 74% yield.
Alkylation of N,N′-dibenzyl-1,3-diaminopropane
(DBnDAP) with chiral epoxide 17 (2.0 equiv) previously
resulted in fully N-protected (2S,10S)-(HO)2DENSPM
in 80% yield.30 Heating only 0.6 equiv of electrophile
17 with DBnDAP in EtOH led to 1:1 adduct 18 as the
major product (64%). The secondary amino function of
18 added efficiently to epoxide 16 at its less substituted
carbon, affording masked tetraamine 19 (94%). Depro-
tection of 19 to analogue 2 was achieved in two stages.
Removal of the trifluoromethanesulfonyls from 19 was
accomplished with Red-Al in toluene at reflux,38 fur-
nishing (2S,10S)-N1-cyclopropylmethyl-N4,N8-dibenzyl-
2,10-dihydroxy-N11-ethylnorspermine (20) in 60% yield.
Hydrogenation (1 atm) in 1 M HCl (8 equiv) and EtOH
over 10% Pd-C removed the N-benzyl moieties of 20,
giving (2R,10S)-(HO)2CPMENSPM (2) as its tetrahy-
drochloride salt in 70% recrystallized yield. The overall
yield was 8% for unsymmetrically dialkylated, outer-
chain-dihydroxylated norspermine analogue 2.
The key reaction in the preparation of (2R,10S)-
(HO)2CPMENSPM (2) was alkylation of norspermidine
derivative 18 with N-cyclopropylmethyl-N-[(2S)-2,3-
epoxypropyl]trifluoromethanesulfonamide (16), which
was prepared according to the method of N-ethylamine-
derived epoxide 17.30 The cyclopropyl terminus of
analogue 2 originated from conversion of cyclopropyl-
methylamine hydrochloride (6) to N-cyclopropylmeth-
yltriflamide (12) in 50% distilled yield using trifluo-
romethanesulfonic anhydride (NEt3/CH2Cl2) (Scheme 3).
Sulfonamide 12 was alkylated with (S)-(+)-2,2-dimethyl-
1,3-dioxolane-4-methanol under Mitsunobu conditions
(diisopropyl azodicarboxylate, triphenylphosphine, THF)
to generate adduct 13 in 96% yield. Cleavage of the ketal

2454 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Bergeron et al.
Ta ble 3. Impact of DENSPM, Dihydroxylated DENSPMs,
CPMENSPM, and (2R,10S)-(HO)2CPMENSPM on Ornithine
Decarboxylase (ODC), S-Adenosylmethionine Decarboxylase
(AdoMetDC), and Spermidine/Spermine-N1-acetyltransferase
(SSAT) in L1210 Cellsa
Ta ble 1. L1210 Cell Growth Inhibition and Transport for
DENSPM, Dihydroxylated DENSPMs, CPMENSPM, and
(2R,10S)-(HO)2CPMENSPM
IC50a (µM)
compound abbreviation
48 h
96 h
Kib (µM)
compound
ODC
AdoMetDC
SSAT
DENSPMc
20
2.0
2.0
0.6
0.55
0.97
17
68
43
13
40
DENSPM
7b
35
61
26
37
42b
59
51
48
59
3877
177
816
3258
2497
(2R,10R)-(HO)2DENSPMc
(2S,10S)-(HO)2DENSPMc
CPMENSPM
3.0
1.0
16
(2R,10R)-(HO)2DENSPMb
(2S,10S)-(HO)2DENSPMb
CPMENSPM
(2R,10S)-(HO)2CPMENSPM
4.5
(2R,10S)-(HO)2CPMENSPM
a
The IC50 was estimated from growth curves for L1210 cells
grown in the presence of nine different concentrations of drug
spanning four logarithmic units: 0, 0.03, 0.1, 0.3, 1.0, 3, 10, 30,
and 100 µM. IC50 data are presented as the mean of at least two
experiments with variation from the mean typically 10-25% for
a
Enzyme activity is expressed as percent of untreated control
for ODC (1 µM at 4 h), AdoMetDC (1 µM at 6 h), and SSAT (10
µM at 48 h for all of the above analogues except DENSPM, which
is 2 µM). Each experiment included a positive control, which had
a known, reproducible impact on enzyme activities (mean ( SD):
1 µM DEHSPM lowered ODC to 6.7 ( 2.6% of untreated control;
1 µM DEHSPM decreased AdoMetDC to 40.7 ( 6.2% of untreated
control; and 2 µM DENSPM increased SSAT to 3877 ( 76% of
untreated control. Data shown in the table represent the mean of
at least three experiments and have variances consistent with
those suggested by the positive control data presented above.
b
the 96-h IC50 values. Ki determinations were made by following
analogue inhibition of spermidine transport. All polyamine ana-
logues exhibited simple substrate-competitive inhibition of [3H]SPD
transport by L1210 cells. Values reported in the table represent
the mean of at least two or three experiments with a variation
typically <10%. c These results are from ref 30.
b
These results are from ref 30.
Ta ble 2. Effect of DENSPM, Dihydroxylated DENSPMs,
CPMENSPM, and (2R,10S)-(HO)2CPMENSPM on Polyamine
Pools in L1210 Cells
Ta ble 4. Survival of CD-1 Mice Treated with DENSPM,
(R,R)-(HO)2DENSPM, CPMENSPM, or
concn
(2R,10S)-(HO)2CPMENSPM under an Acute Toxicity Regimena
compound
DENSPMc
(µM) PUTa SPDa SPMa analogueb
dose
(mg/kg)
no. of deaths/no. of
mice injected
10
100
3
15
1
30
0
14
6
31 1.59
30 2.44
compound
DENSPMb
(2R,10R)-(HO)2DENSPM
CPMENSPM
(2R,10R)-(HO)2DENSPMc
(2S,10S)-(HO)2DENSPMc
CPMENSPM
9
5
14
9
63 1.88
54 1.99
325
325
100
200
100
200
400
5/10
0/6
1/5c
5/5c
0/5
66
5
46
7
94 1.18
56 2.02
5
CPMENSPM
20
100
4
22
13
43
20
25
16
48
32
66 2.17 ( 0.11
56 2.59 ( 0.19
119 1.94 ( 0.03
123 3.05 ( 0.37
(2R,10S)-(HO)2CPMENSPM
(2R,10S)-(HO)2CPMENSPM
(2R,10S)-(HO)2CPMENSPM
0/5
0/5d
(2R,10S)-(HO)2CPMENSPM
20
a
The polyamine analogues were administered to female CD-1
mice at the doses indicated as a single ip injection. The mice were
monitored for 2 h after administration of drug, after which scoring
took place. Their overall health was then checked daily during a
a
Putrescine (PUT), spermidine (SPD), and spermine (SPM)
levels after 48 h of treatment are given as percent of the polyamine
found in untreated controls. Typical control values in pmol/106
L1210 cells are PUT ) 260 ( 59, SPD ) 3354 ( 361, and SPM )
b
10-day postdosing observational period. These results are from
b
658 ( 119. Analogue amount is expressed as nmol/106 cells.
ref 21. c Those animals that died did so in a matter of minutes of
convulsions and asphyxiation; all mice exhibited pronounced signs
of CNS toxicity, including splayed hind limbs, intention tremors,
and ataxia. The survivors appeared to be healthy throughout the
Untreated L1210 cells (106) correspond to ∼1 µL volume; therefore,
the concentration can be estimated as mM. c These results are
from ref 30.
d
10-day postdosing observational period. These animals exhibited
significant CNS signs during the first several hours after injection
but had recovered completely by the next morning.
Biologica l Eva lu a tion s
The biological studies encompass both in vitro and in
vivo assessments of the polyamine analogues. In each
instance, the findings are compared to historical data
for DENSPM and its hydroxylated counterparts, (2R,-
10R)-(HO)2DENSPM and (2S,10S)-(HO)2DENSPM.30
The in vitro measurements, conducted in L1210 cells,
include IC50 and Ki determinations (Table 1), as well
as evaluations of how the analogues accumulate intra-
cellularly (Table 2), affect the native polyamine pools
(Table 2), and impact the polyamine enzymes ODC,
AdoMetDC, and SSAT (Table 3). The in vivo assessment
involves a comparison of the acute and chronic toxicities
(Tables 4 and 5, respectively) of the parent and hy-
droxylated compounds in mice.
IC50 Mea su r em en ts. As with DENSPM (20 µM) and
its hydroxylated analogues [(2R,10R)-(HO)2DENSPM,
3.0 µM, and (2S,10S)-(HO)2DENSPM, 1.0 µM] at 48 h,
the hydroxylated analogue of CPMENSPM, (2R,10S)-
(HO)2CPMENSPM (4.5 µM), was more active than its
parent (16 µM) (Table 1). At 96 h, again in a manner
similar to that of the DENSPMs, the large differences
in IC50 values between hydroxylated and parent drug
were absent.
Ki Stu d ies. The ability of CPMENSPM and (2R,10S)-
(HO)2CPMENSPM to compete with radiolabeled sper-
midine for the polyamine transport apparatus (Ki) was
determined. In accord with the results for (2R,10R)-
(HO)2DENSPM (68 µM) and (2S,10S)-(HO)2DENSPM
(43 µM) versus that for DENSPM (17 µM), the Ki value
for (2R,10S)-(HO)2CPMENSPM (40 µM) was also higher
than that of its parent drug, CPMENSPM (13 µM). The
most likely explanation is that hydroxylated norsper-
mines simply do not bind as well to the polyamine
transport apparatus as do the nonhydroxylated com-
pounds.
Im p a ct of An a logu es on P olya m in e P ools. The
intracellular accumulations of CPMENSPM and (2R,-
10S)-(HO)2CPMENSPM as well as the impact of these
compounds on polyamine pools in L1210 cells were
measured after 48 h of incubation. Two different con-
centrations were used, the approximate 48-h IC50
concentration and 5 times this value (Table 2). In the
case of DENSPM and its hydroxylated analogues, at
either concentration assessed, similar intracellular ac-
cumulations of the compound generated comparable

Hydroxylated Polyamine Analogues as Antiproliferatives
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2455
Ta ble 5. Survival of CD-1 Mice Treated with a 5-Day Course
of Hydroxylated versus Nonhydroxylated Norspermine
Analoguesa
neither (2R,10R)-(HO)2DENSPM (177%) nor (2S,10S)-
(HO)2DENSPM (816%) achieved this level of stimula-
tion, even at 5 times this concentration. In contrast, the
increase in SSAT activity elicited by CPMENSPM
(3258% of control value) is analogous to that observed
with DENSPM; however, unlike either of the hydroxy-
lated DENSPMs, (2R,10S)-(HO)2CPMENSPM increased
SSAT activity by 2497% of control value, a level
comparable to that of its parent drug.
dose
no. of no. of deaths/no.
compound
(mg/kg/day) days of mice injected
DENSPMb
200
217
10
25
50
6
6
5
5
5
5
5
5
5
5
10/12
0/15
0/7
0/7
0/7
(2R,10R)-(HO)2DENSPMc
CPMENSPM
75
0/5
0/5d
0/6
Toxicit y of CP ME NSP M a n d (2R,10S)-(H O)2-
CP MENSP M in Mice. The toxicity of CPMENSPM is
acute in nature, including a strong neurological com-
ponent. All mice treated with CPMENSPM at an ip dose
of g100 mg/kg exhibited pronounced signs of central
nervous system (CNS) toxicity. These included an ataxic
gait, intention tremors, and severe motor dysfunction,
especially of the hindlimbs (Table 4). Those animals that
died did so in a matter of minutes of convulsions and
asphyxiation. The survivors appeared to be healthy
throughout the 10-day postdosing observational period.
When animals were treated in a chronic toxicity protocol
(daily ip injections for 5 days), the only signs of toxic-
ity were observed immediately after injection. Again,
these consisted of acute CNS signs, which were moder-
ate at 75 mg/kg and severe at 100 mg/kg. No signs of
chronic toxic effects, such as weight loss or poor coat
health, were observed during the treatment period or
during a 10-day post-treatment followup (Table 5). In
marked contrast, only minimal toxic effects (ataxia of
15-20-min duration) were observed in the five mice
treated with (2R,10S)-(HO)2CPMENSPM at 200 mg/kg,
a dose 93% on a molar basis of that which was lethal to
five of five mice treated with CPMENSPM (Table 4).
Moreover, even at a dose of 400 mg/kg of (2R,10S)-
(HO)2CPMENSPM, there were no deaths among the five
mice injected, although there were significant neuro-
logical signs at this dose. Even when the mice received
(2R,10S)-(HO)2CPMENSPM at doses of 100, 200, or 300
mg/kg/day under a 5-day chronic dosing regimen, there
was no apparent toxicity beyond what was found during
the acute study. In fact, the neurological signs that were
observed in the mice treated with the hydroxylated
analogue at 300 mg/kg/day were much milder than
those in the animals given the nonhydroxylated com-
pound at approximately one-third the dose (Table 5).
Thus, the introduction of hydroxyls reduced the toxicity
at least severalfold.
100
100
200
300
(2R,10S)-(HO)2CPMENSPM
0/6
0/5e
a
Female CD-1 mice were administered the polyamine analogues
as 1 ip dose/day for the number of days indicated. Animals were
b
monitored daily for an additional 10 days. These results are from
ref 21. During the course of treatment, all of the animals displayed
severe neurological signs, which included severe ataxia, intention
tremors, and motor dysfunction. By day 3, two of the animals had
died of seizures; by the end of the dosing period, the number of
animals indicated had died from neurological sequelae. c The data
are from ref 30. Neither weight loss nor any other adverse effects
d
were observed. Animals in the 100 mg/kg group exhibited
pronounced signs of CNS toxicity, such as splayed hind limbs,
tremors, and ataxia, immediately after the dose, but the mice
recovered after several hours. No signs of chronic toxicity, such
as weight loss or changes in coat appearance, were observed during
the 5-day treatment period; all mice appeared to be healthy
throughout the 10-day postdosing observational period. e Animals
in the 300 mg/kg group exhibited mild signs of CNS toxicity, such
as ataxia, immediately after the dose, but the mice completely
recovered after several hours. No signs of chronic toxicity, such
as weight loss or changes in coat appearance, were observed during
the 5-day treatment period; all mice appeared to be healthy
throughout the 10-day postdosing observational period.
impacts on polyamine pools. This was not quite the case
with CPMENSPM and its hydroxylated counterpart. At
its 48-h IC50 concentration, CPMENSPM diminished the
putrescine (PUT), spermidine (SPD), and spermine
(SPM) levels to 22, 25, and 66%, respectively, of control
values; CPMENSPM reached an intracellular concen-
tration of 2.17 ( 0.11 mM. At its approximate 48-h IC50
concentration (4 µM), (2R,10S)-(HO)2CPMENSPM at-
tained a concentration of 1.94 ( 0.03 mM and concomi-
tantly lowered PUT and SPD to 43 and 48%, respec-
tively, of controls, but SPM was slightly elevated (119%
of control value). The reduction of polyamine pools was
not as significant with (2R,10S)-(HO)2CPMENSPM,
particularly considering that SPM was unaffected or
slightly enhanced at both drug concentrations tested.
Effect of Com p ou n d s on ODC, Ad oMetDC, a n d
SSAT. In each case, ODC and AdoMetDC measure-
ments were made in L1210 cells after exposure to 1 µM
polyamine analogue. Unlike DENSPM (7% of control
value) and its hydroxylated analogues [(2R,10R)-, 35%
of control value; (2S,10S)-, 61%, Table 3], there was not
a substantial difference in ODC activity after treatment
with (2R,10S)-(HO)2CPMENSPM (37%) or CPMENSPM
(26%). However, the moderate impact of both
CPMENSPM (48% of control value) and (2R,10S)-(HO)2-
CPMENSPM (59%) on AdoMetDC resembled that of
DENSPM (42%) and its hydroxylated derivatives
[(2R,10R)-, 59% of control value; (2S,10S)-, 51%].
The most notable difference between the DENSPMs
and the CPMENSPMs is related to their stimulation
of SSAT after a 48-h incubation with 10 µM poly-
amine analogue. Whereas DENSPM dramatically up-
regulated SSAT (3877% of control value) at 2 µM,
Discu ssion
The current synthetic route to CPMENSPM (1) offers
a number of advantages over the previously described
approach.31 The scheme is shorter (8 versus 10 steps)
and generates a higher yield (10 versus 2%), offering
facile access to crystalline product. The key step in-
volved alkylation of the anion of N,N′-bis(mesitylene-
sulfonyl)-N-cyclopropylmethyl-1,3-diaminopropane with
N,N′-bis(mesitylenesulfonyl)-N-(3-bromopropyl)-N′-ethyl-
1,3-diaminopropane, followed by removal of the mes-
itylenesulfonamide protecting groups.
The seminal step in the assembly of the hydroxylated
analogue (2) required two different alkylating agents,
N-[(2S)-2,3-epoxypropyl]-N-ethyltrifluoromethanesulfon-
amide and N-cyclopropylmethyl-N-[(2S)-2,3-epoxypro-
pyl]trifluoromethanesulfonamide. The reagents were

2456 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Bergeron et al.
(2 × 200 mL). The combined organic layers were washed with
0.5 N HCl (200 mL) and 1:1 H2O/brine (200 mL) and were
concentrated in vacuo. Recrystallization from aqueous EtOH
gave 80.57 g of 4 (88%) as a solid: mp 103.5-104 °C; 1H NMR
δ 0.95 (t, 3 H, J ) 7.2), 1.68 (quintet, 2 H, J ) 6.6), 2.30 (s, 6
H), 2.56 (s, 6 H), 2.62 (s, 6 H), 2.92 (quartet, 2 H, J ) 6.6),
3.12 (quartet, 2 H, J ) 7.2), 3.31 (t, 2 H, J ) 6.6), 4.92 (t, 1 H,
J ) 6.6), 6.94 (s, 2 H), 6.95 (s, 2 H); 13C NMR δ 12.51, 20.84,
22.74, 22.82, 27.73, 39.23, 39.94, 42.23, 131.87, 131.97, 133.20,
133.94, 138.82, 139.91, 141.95, 142.50; HRMS, m/z calcd for
coupled in a stepwise fashion to N,N′-dibenzyl-1,3-
diaminopropane, and the triflyl and benzyl protecting
groups were removed. Once again, the chemistry is
relatively simple (nine steps) and effective, accomplished
in nearly the same overall yield (8%) as that of (2S,-
10S)-(HO)2DENSPM (10%),30 despite potential stability
problems with the cyclopropyl ring39,40 in 2.
In a comparison of how hydroxylation of DENSPM
and CPMENSPM influences their respective biological
properties, the similarities are far more common than
the differences. In both instances, hydroxylation has
only a minor effect on the 96-h IC50 values, and the Ki
values are increased. Regarding the impact of the
analogues on polyamine pools, when similar intracel-
lular levels of drug are considered, hydroxylation exerts
little effect on DENSPM’s reduction of polyamine pools.
Although hydroxylation of CPMENSPM moderately
reduces the molecule’s ability to diminish PUT and SPD,
this modification significantly alters CPMENSPM’s
depletion of SPM. Hydroxylation of DENSPM also
attenuates its ability to decrease ODC and AdoMetDC
as well as its capability to stimulate SSAT. This was
not the case with CPMENSPM hydroxylation. There
was little change in ODC and AdoMetDC and only a
very modest effect on SSAT stimulation.
With both DENSPM and CPMENSPM, the most
interesting observation was that hydroxylation pro-
foundly reduced the compounds’ neurologic toxicity.
Thus, hydroxylation of norspermine analogues may be
a means for increasing their therapeutic indices. How-
ever, the question regarding the generality of this
approach remains to be answered. In particular, as the
lipophilicity of the molecule increases (i.e., by adding
larger terminal alkyl groups), will it be necessary to
likewise increase the number of appended hydroxyls to
reduce toxicity? Experiments designed to define the
possible relationships among hydrophobicity, the ability
to penetrate the blood-brain barrier, and CNS toxicity
of norspermine analogues are ongoing.
C
23H35N2O4S2 467.2038 (M + H), found 467.2057. Anal. Calcd
(C23H34N2O4S2) C, H, N.
N,N′-Bis(m esit ylen esu lfon yl)-N-(3-b r om op r op yl)-N′-
eth yl-1,3-d ia m in op r op a n e (5b). Sodium hydride (60%, 2.23
g, 55.7 mmol) was added to 4 (20.0 g, 42.9 mmol) in DMF (300
mL) and stirred for 30 min in an ice bath and for 30 min at
room temperature. The solution was added slowly to 1,3-
dibromopropane (104 g, 0.514 mol) in DMF (200 mL). The
mixture was stirred for 18 h at room temperature. The reaction
was quenched with EtOH (10 mL) and H2O (20 mL) after 40
h, and solvents were removed in vacuo. The residue was
dissolved in CHCl3 (200 mL), washed with brine (200 mL), and
concentrated in vacuo. Flash chromatography (15-33% EtOAc/
hexane) afforded 17.8 g of 5b (71%) as a white glassy solid:
1H NMR δ 1.00 (t, 3 H, J ) 7.2), 1.71 (quintet, 2 H, J ) 7.2),
1.96 (quintet, 2 H, J ) 7.2), 2.30 (s, 6 H), 2.56 (s, 6 H), 2.57 (s,
6 H), 3.01-3.09 (m, 4 H), 3.14 (quartet, 2 H, J ) 7.2), 3.21-
3.28 (m, 4 H), 6.93 (s, 2 H), 6.94 (s, 2 H); 13C NMR δ 12.67,
20.91, 22.72, 22.77, 25.43, 30.18, 30.38, 40.12, 42.54, 43.81,
44.35, 131.90, 132.01, 132.71, 133.09, 140.04, 140.09, 142.37,
142.68; HRMS, m/z calcd for C26H40BrN2O4S2 587.1613 (M +
H), found 587.1603.
N-Cyclop r op ylm eth ylm esitylen esu lfon a m id e (7). Mes-
itylenesulfonyl chloride (22.4 g, 0.102 mol), 6 (10.0 g, 0.093
mol), and 1 N NaOH (250 mL) were reacted according to the
method of 4. Recrystallization of the crude product from EtOH
1
gave 23.0 g of 7 (97%) as a solid: mp 69-70 °C; H NMR δ
0.04-0.11 (m, 2 H), 0.42-0.50 (m, 2 H), 0.79-0.94 (m, 1 H),
2.30 (s, 3 H), 2.65 (s, 6 H), 2.76 (dd, 2 H, J ) 6.9, 6.0), 4.57 (br
s, 1 H), 6.96 (s, 2 H); 13C NMR δ 3.45, 10.59, 20.89, 22.89, 47.76,
131.87, 133.69, 139.00, 142.05; HRMS, m/z calcd for C13H20
NO2S 254.1215 (M + H), found 254.1198. Anal. Calcd (C13H19
NO2S) C, H, N.
-
-
N-Cyclop r op ylm et h yl-N-(3-p h t h a lim id op r op yl)m es-
itylen esu lfon a m id e (8). Sodium hydride (60%, 4.74 g, 0.118
mol) was added to 7 (20.0 g, 78.9 mmol) in DMF (300 mL)
under ice-bath conditions. The mixture was stirred for 30 min
and then at room temperature for an additional 30 min. N-(3-
Bromopropyl)phthalimide (31.7 g, 0.118 mol) was added fol-
lowed by heating at 40 °C for 4 h. The reaction was quenched
with EtOH (10 mL) and H2O (20 mL), and solvents were
removed in vacuo. The residue was dissolved in CHCl3 (1 L)
and washed with H2O (500 mL). Flash chromatography (4%
acetone/toluene) gave 29.2 g of 8 (84%) as a white solid: mp
93-96 °C; 1H NMR δ 0.08-0.16 (m, 2 H), 0.45-0.53 (m, 2 H),
0.85-1.00 (m, 1 H), 1.84 (quintet, 2 H, J ) 7.5), 2.25 (s, 3 H),
2.55 (s, 6 H), 3.12 (d, 2 H, J ) 7.2), 3.32 (t, 2 H, J ) 7.5), 3.54
(t, 2 H, J ) 7.5), 6.83 (s, 2 H), 7.70-7.78 (m, 2 H), 7.80-7.88
(m, 2 H); 13C NMR δ 3.93, 9.31, 20.92, 22.68, 26.51, 35.33,
43.32, 50.13, 123.17, 131.80, 131.92, 132.79, 133.96, 140.08,
142.31, 168.06; HRMS, m/z calcd for C24H29N2O4S 441.1848
(M + H), found 441.1816.
N-(3-Am in op r op yl)-N-cyclop r op ylm et h ylm esit ylen e-
su lfon a m id e (9). Hydrazine hydrate (55%, 3.46 g, 59.4 mmol)
and 8 (11.9 g, 27.0 mmol) were heated at reflux in EtOH (270
mL) for 12 h, the mixture was filtered through Celite, and
solvents were removed in vacuo. Flash chromatography (2%
concentrated NH4OH/CH3OH) afforded 7.3 g of 9 (87%) as a
thick oil: 1H NMR δ 0.03-0.10 (m, 2 H), 0.43-0.51 (m, 2 H),
0.79-0.93 (m, 1 H), 1.70 (quintet, 2 H, J ) 7), 1.86 (br s, 2 H),
2.29 (s, 3 H), 2.60 (s, 6 H), 2.65 (t, 2 H, J ) 7), 3.03 (d, 2 H, J
) 7), 3.40 (t, 2 H, J ) 7), 6.93 (s, 2 H); 13C NMR δ 3.83, 8.99,
20.90, 22.75, 30.57, 39.08, 43.10, 49.84, 131.84, 133.24, 140.11,
Exp er im en ta l Section
Gen er a l. Compound 3 was obtained from Karl Industries,
Aurora, OH. Other reagents were purchased from the Aldrich
Chemical Co., Milwaukee, WI. Fisher Optima-grade solvents
were routinely used, and organic extracts were dried with
sodium sulfate. DMF and THF were distilled, the latter from
sodium and benzophenone. Reactions in nonaqueous solvents
were run under a nitrogen atmosphere. Silica gel 32-63 from
Selecto Scientific, Inc. (Suwanee, GA) was used for flash
column chromatography. Melting points are uncorrected. NMR
spectra were recorded at 300 MHz (1H) or at 75 MHz (13C) on
a Varian Unity 300. Compounds 1 and 2 were measured in
D2O with sodium 2,2,3,3-d4-3-(trimethylsilyl)propionate (TSP,
δ 0.0) for 1H or dioxane (δ 67.19) for 13C as references. Other
spectra were run in CDCl3 with tetramethylsilane (δ 0.0) for
1H or the solvent (δ 77.0) for 13C as standards. Coupling
constants (J ) are in hertz. Optical rotations were measured
in CHCl3 (unless specified) at 589 nm (sodium D line) with a
Perkin-Elmer 341 polarimeter; c equals grams of compound
per 100 mL of solution. FAB mass spectra were run in a
3-nitrobenzyl alcohol or glycerol (1 and 2) matrix. Elemental
analyses were performed by Atlantic Microlabs, Norcross, GA.
N,N′-Bis(m esitylen esu lfon yl)-N-eth yl-1,3-d ia m in op r o-
p a n e (4). A solution of mesitylenesulfonyl chloride (85.43 g,
0.391 mol) in CH2Cl2 (260 mL) was added to 3 (20.07 g, 0.196
mol) in 2 N NaOH (210 mL) with ice-bath cooling. The mixture
was stirred for 18 h at room temperature, the layers were
separated, and the aqueous portion was extracted with CHCl3

Hydroxylated Polyamine Analogues as Antiproliferatives
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2457
142.25; HRMS, m/z calcd for C16H27N2O2S 311.1793 (M + H),
room temperature, and the reaction mixture was stirred
overnight. The solvent was removed in vacuo, and the crude
product was purified by flash chromatography (CHCl3) to give
6.10 g of 13 (96%) as a colorless oil: 1H NMR δ 0.27 (m, 1 H),
0.35 (m, 1 H), 0.60 (m, 2 H), 1.04 (m, 1 H), 1.32 (s, 3 H), 1.40
(s, 3 H), 3.43 (m, 1 H), 3.51 (dd, 2 H, J ) 15.0, 7.2), 3.65 (dd,
1 H, J ) 8.7, 6.3), 3.69 (m, 1 H), 4.08 (dd, 1 H, J ) 8.7, 6.3),
4.31 (m, 1 H); 13C NMR δ 3.39, 4.58, 9.21, 25.25, 26.67, 54.22,
67.16, 75.26, 77.20, 110.06, 120.01 (q, J ) 328); HRMS, m/z
calcd for C11H19F3NO4S 318.0987 (M + H), found 318.0985;
found 311.1776.
N,N′-Bis(m esit ylen esu lfon yl)-N-cyclop r op ylm et h yl-
1,3-d ia m in op r op a n e (10). Mesitylenesulfonyl chloride (7.75
g, 35.4 mmol), 9 (10.0 g, 32.2 mmol), and 1 N NaOH (50 mL)
were reacted according to the method of 4. Flash chromatog-
raphy (8% acetone/toluene) generated 13.4 g of 10 (84%) as a
1
white solid: mp 92-94 °C; H NMR δ -0.06 to +0.05 (m, 2
H), 0.39-0.48 (m, 2 H), 0.67-0.80 (m, 1 H), 1.72 (quintet, 2
H, J ) 6.3), 2.30 (s, 6 H), 2.57 (s, 6 H), 2.62 (s, 6 H), 2.87-2.96
(m, 4 H), 3.45 (t, 2 H, J ) 6.3), 4.95 (t, 1 H, J ) 6.3), 6.93 (s,
2 H), 6.95 (s, 2 H); 13C NMR δ 3.85, 8.89, 20.94, 22.79, 22.87,
27.71, 39.24, 42.93, 50.01, 131.91, 132.00, 133.15, 134.02,
[R]25 -20.1 (c 2.33).
D
N-Cyclop r opylm eth yl-N-[(2S)-2,3-dih ydr oxyp r op yl]tr i-
flu or om eth a n esu lfon a m id e (14). A solution of 13 (5.91 g,
18.6 mmol) in acetone (40 mL) and 1 N HCl (80 mL) was
heated at reflux for 2 h. The solvent was removed in vacuo,
and the residue was purified by flash chromatography (15:1
CHCl3/CH3OH) to give 5.07 g of 14 (98%) as an oil: 1H NMR
δ 0.24 (m, 2 H), 0.57 (m, 2 H), 0.97 (m, 1 H), 2.60 (s, 2 H), 3.30
(s, 2 H), 3.52 (m, 3 H), 3.67 (dd, 1 H, J ) 11.7, 3.6), 3.93 (m,
1 H); 13C NMR δ 4.11, 9.42, 50.00, 54.69, 63.54, 70.03, 120.05
(q, J ) 323); HRMS, m/z calcd for C8H15F3NO4S 278.0674 (M
+ H), found 278.0673; [R]2D4 -4.94 (c 1.74).
138.87, 140.05, 141.97, 142.56; HRMS, m/z calcd for C25H37
N2O4S2 493.2195 (M + H), found 493.2132.
-
N1-Cyclop r op ylm eth yl-N11-eth yl-N1,N4,N8,N11-tetr a k is-
(m esitylen esu lfon yl)n or sp er m in e (11).31 Sodium hydride
(60%, 0.39 g, 9.8 mmol) was added to 10 (3.7 g, 7.5 mmol) in
DMF (50 mL) under ice-bath conditions. The mixture was
stirred for an additional 30 min each at 0 °C and at room
temperature and was added dropwise to 5b (5.3 g, 9.0 mmol)
in DMF (75 mL). The mixture was stirred overnight at room
temperature and was quenched under ice-bath conditions with
EtOH (10 mL) and H2O (10 mL). The solvents were removed
in vacuo, and the concentrate was taken up in CHCl3 (50 mL)
and H2O (50 mL). The chloroform layer was washed with brine
(50 mL); the solution was concentrated in vacuo. Flash
chromatography (3:1 hexane/EtOAc) gave 5.47 g of 10 (73%)
as a glassy white solid: 1H NMR δ -0.08 to -0.02 (m, 2 H),
0.38-0.45 (m, 2 H), 0.64-0.78 (m, 1 H), 0.95 (t, 3 H, J ) 7.2),
1.56-1.73 (m, 6 H), 2.29 (s, 12 H), 2.53 (s, 12 H), 2.54 (s, 12
H), 2.86 (d, 2 H, J ) 6.6), 2.91-3.17 (m, 14 H), 6.93 (s, 8 H);
13C NMR δ 3.77, 8.90, 12.61, 20.90, 22.68, 22.69, 22.74, 22.76,
25.08, 25.18, 25.28, 40.04, 42.49, 43.20, 49.92, 131.88, 131.96,
132.91, 132.96, 133.02, 133.10, 140.00, 140.02, 140.03, 142.32,
142.49, 142.52; HRMS, m/z calcd for C51H75N4O8S4 999.4468
(M + H), found 999.4382.
N-Cyclop r op ylm eth yl-N-[(2S)-2-h yd r oxy-3-(p-tolu en e-
su lfon a to)p r op yl]tr iflu or om eth a n esu lfon a m id e (15). p-
Toluenesulfonyl chloride (3.70 g, 19.4 mmol) in anhydrous
CH
2Cl2 (20 mL) was added dropwise to 14 (4.89 g, 17.7 mmol)
in anhydrous pyridine (25 mL) at 0 °C. After the reaction
mixture had been stirred at room temperature overnight, it
was poured into a slurry of 1 N HCl (300 mL) and ice and
then extracted with CHCl3 (300 mL). The organic layer was
washed with H2O (300 mL) and brine (300 mL), concentrated
under reduced pressure, and purified by flash chromatography
(40:1 CHCl3/CH3OH) to give 6.70 g of 15 (88%) as an oil: 1H
NMR δ 0.28 (m, 2 H), 0.61 (m, 2 H), 0.98 (m, 1 H), 2.44 (s, 3
H), 2.63 (br, 1 H), 3.33 (m, 2 H), 3.49 (dd, 1 H, J ) 15.3, 7.8),
3.58 (dd, 1 H, J ) 15.3, 4.2), 4.02 (m, 2 H), 4.14 (m, 1 H), 7.35
(d, 2 H, J ) 8.1), 7.78 (d, 2 H, J ) 8.1); 13C NMR δ 3.96, 4.27,
9.32, 21.65, 49.94, 54.90, 68.48, 71.03, 119.97 (q, J ) 323),
127.96, 130.06, 132.09, 145.47; HRMS, m/z calcd for C15H21F3-
NO6S2 432.0762 (M + H), found 432.0765; [R]2D2 -3.83 (c
1.07).
N1-Cyclop r op ylm eth yl-N11-eth yln or sp er m in e Tetr a h y-
d r och lor id e (1).31 Hydrogen bromide in HOAc (30%, 50 mL)
was added to 11 (5.3 g, 5.3 mmol) and phenol (19.9 g, 0.212
mol) in CH2Cl2 (50 mL) at 0 °C. The reaction mixture was
stirred overnight at room temperature and was quenched with
H2O (100 mL) under ice-bath conditions. The aqueous layer
was washed with CH2Cl2 (4 × 100 mL), and the former was
concentrated in vacuo. The residue was treated with 1 N
NaOH (20 mL) and 19 N NaOH (10 mL) under ice-bath cooling
and was extracted with CHCl3 (4 × 50 mL). After concentration
of the CHCl3 extracts, the oil was dissolved in EtOH (50 mL)
and acidified with concentrated HCl (10 mL). Solvent removal
in vacuo followed by recrystallization from aqueous EtOH gave
N-Cyclop r op ylm eth yl-N-[(2S)-2,3-ep oxyp r op yl]tr iflu o-
r om eth a n esu lfon a m id e (16). A suspension of 15 (6.59 g,
15.3 mmol) and K2CO3 (2.24 g, 16.2 mmol) in CH3OH (100 mL)
was stirred at room temperature for 4 h. Solids were filtered,
and brine (100 mL), water (150 mL), and CH2Cl2 (150 mL)
were added to the filtrate. The layers were separated, and the
aqueous layer was extracted with CH2Cl2 (2 × 100 mL). The
organic phase was evaporated under reduced pressure and
purified by flash chromatography (4:1 cyclohexane/EtOAc) to
give 2.93 g of 16 (74%) as an oil: 1H NMR δ 0.27 (m, 1 H),
0.35 (m, 1 H), 0.60 (m, 2 H), 1.07 (m, 1 H), 2.56 (dd, 1 H, J )
4.5, 2.4), 2.82 (t, 1 H, J ) 4.5), 3.16 (m, 1 H), 3.31 (m, 2 H),
3.43 (m, 1 H), 3.95 (dd, 1 H, J ) 15.0, 3.0); 13C NMR δ 3.49,
4.52, 9.45, 44.67, 50.54, 54.22, 119.99 (q, J ) 323); HRMS,
m/z calcd for C8H13F3NO3S (M + H), 260.0568, found 260.0560;
[R]2D2 -32.1 (c 1.00).
1
0.800 g of 1 (36%) as white flakes: mp >290 °C; H NMR δ
0.34-0.41 (m, 2 H), 0.66-0.74 (m, 2 H), 1.02-1.17 (m, 1 H),
1.30 (t, 3 H, J ) 7.5), 2.07-2.21 (m, 6 H), 2.98 (d, 2 H, J )
7.5), 3.08-3.26 (m, 14 H); 13C NMR δ 4.04, 7.22, 11.14, 23.34,
43.71, 44.47, 45.22, 45.31, 53.29; HRMS, m/z calcd for C15H35N4
271.2862 (M + H), found 271.2861. Anal. Calcd (C15H38Cl4N4)
C, H, N.
(2R)-N4,N7-Dib en zyl-N1-et h yl-2-h yd r oxy-N1-t r iflu or o-
m eth a n esu lfon yln or sp er m id in e (18). A solution of 17
(11.36 g, 50.89 mmol)30 and N,N′-dibenzyl-1,3-diaminopropane
(21.80 g, 85.70 mmol)42 in EtOH (200 mL) was heated at reflux
overnight, and the solvent was removed under reduced pres-
sure. The residue was purified using flash chromatography
(60:39.5:0.5 toluene/acetone/concentrated NH4OH) to afford
15.80 g of 18 (64%) as an oil: 1H NMR δ 1.13 (t, 3 H, J ) 7.2),
1.64 (m, 2 H), 2.37 (m, 2 H), 2.52 (m, 1 H), 2.65 (m, 3 H), 3.17
(m, 2 H), 3.40 (d, 1 H, J ) 13.5), 3.46 (m, 2 H), 3.64 (d, 1 H, J
) 13.5), 3.68 (s, 2 H), 3.73 (m, 1 H), 5.31 (br s, 1 H), 7.20 (m,
10 H); 13C NMR δ 13.49, 26.86, 44.80, 47.06, 51.19, 52.24,
53.97, 57.63, 59.46, 68.21, 120.00 (q, J ) 324), 127.03, 127.21,
128.28, 128.37, 128.41, 129.00, 138.64, 139.73; HRMS, m/z
calcd for C23H33F3N3O3S 488.2195 (M + H), found 488.2206;
[R]2D4 +12.6 (c 1.00).
N-Cyclop r op ylm et h ylt r iflu or om et h a n esu lfon a m id e
(12). Trifluoromethanesulfonic anhydride (27.00 g, 95.7 mmol)
in CH2Cl2 (50 mL) was added dropwise to 6 (10.30 g, 95.7
mmol) and Et3N (40 mL, 0.29 mol) in CH2Cl2 (100 mL) at -78
°C, and the reaction mixture was stirred overnight at room
temperature. The reaction solution was washed with 1 N HCl
(3 × 100 mL) and brine and was concentrated under reduced
pressure. Vacuum distillation afforded 9.74 g of 12 (50%): bp
1
81-84 °C at 2.6 mmHg; H NMR δ 0.27 (m, 2 H), 0.63 (m, 2
H), 1.08 (m, 1 H), 3.17 (t, 2 H, J ) 6.3), 4.91 (br, 1 H).
N-Cyclopr opylm eth yl-N-[(4S)-2,2-dim eth yl-1,3-dioxolan -
4-ylm eth yl]tr iflu or om eth a n esu lfon a m id e (13). (S)-(+)-
2,2-Dimethyl-1,3-dioxolane-4-methanol (2.64 g, 20.0 mmol) and
triphenylphosphine (6.29 g, 24.0 mmol) were added to 12 (4.06
g, 20.0 mmol) in THF (100 mL). Diisopropyl azodicarboxylate
(4.85 g, 24.0 mmol) in THF (40 mL) was added dropwise at

2458 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Bergeron et al.
(2R,10R)-N1,N11-Bis(tr iflu or om eth a n esu lfon yl)-N1-cy-
clop r op ylm eth yl-N4,N8-d iben zyl-2,10-d ih yd r oxy-N11-eth -
yln or sp er m in e (19). A solution of 16 (2.82 g, 10.9 mmol) and
18 (5.31 g, 10.9 mmol) in EtOH was heated at reflux for 3 days.
The reaction mixture was concentrated under reduced pres-
sure, and the residue was purified by flash chromatography
(3:1 then 1:1 cyclohexane/EtOAc) providing 7.67 g of 19 (94%)
as a viscous oil: 1H NMR δ 0.27 (m, 2 H), 0.54 (m, 2 H), 1.00
(m, 1 H), 1.20 (t, 3 H, J ) 7.2), 1.67 (m, 2 H), 2.39 (m, 6 H),
2.57 (m, 2 H), 3.14 (m, 1 H), 3.38 (m, 7 H), 3.52 (m, 4 H), 3.75
(m, 4 H), 7.17 (m, 4 H), 7.27 (m, 6 H); 13C NMR δ 3.46, 4.37,
9.17, 13.52, 24.27, 45.14, 51.28, 51.82, 54.31, 57.14, 57.30,
58.84, 67.24, 120.02 (q, J ) 320), 127.45, 128.50, 129.09,
137.81; HRMS, m/z calcd for C31H45F6N4O6S2 747.2685 (M +
H), found 747.2687; [R]2D2 +30.5 (c 1.30).
Up ta k e Deter m in a tion s. The polyamine derivatives were
studied for their ability to compete with [3H]SPD for uptake
into L1210 leukemia cell suspensions in vitro as given in detail
in previous publications.12,21,30 Briefly, cell suspensions were
incubated in 1 mL of culture medium containing radiolabeled
SPD alone or radiolabeled SPD in the presence of graduated
concentrations of polyamine analogue. At the end of the
incubation period, the tubes were centrifuged; the pellet was
washed, digested, and neutralized prior to scintillation count-
ing. Lineweaver-Burk plots indicated a simple competitive
inhibition with respect to SPD.
En zym e Assa ys. ODC and AdoMetDC activities were
determined according to the procedures of Seely and Pegg44
and Pegg and Po¨so¨,45 respectively, on the basis of quantitation
of 14CO2 released from [14C]carboxyl-labeled L-ornithine or
S-adenosyl-L-methionine. Included in each assay were un-
treated L1210 cells as negative controls as well as cells treated
with DEHSPM, a drug having a known reproducible effect on
each enzyme, as positive controls.
(2S,10S)-N1-Cyclop r op ylm eth yl-N4,N8-d iben zyl-2,10-d i-
h yd r oxy-N11-eth yln or sp er m in e (20). Red-Al (65% in tolu-
ene, 19.3 mL, 64.2 mmol) was added slowly to a solution of 19
(6.00 g, 8.04 mmol) in toluene (300 mL), and the reaction
mixture was heated at reflux overnight. The reaction mixture
was cautiously quenched with H2O (100 mL) and 10% NaOH
(100 mL) at room temperature. The layers were separated, and
the aqueous portion was extracted with CH2Cl2 (3 × 200 mL).
The combined organic layers were washed with brine (500 mL)
and concentrated under reduced pressure. Purification using
flash chromatography (20:1 and then 10:1 CH3CN/concen-
trated NH4OH) furnished 2.32 g of 20 (60%) as an oil: 1H NMR
δ 0.08 (m, 2 H), 0.44 (m, 2 H), 0.91 (m, 1 H), 1.07 (t, 3 H, J )
7.2), 1.64 (quintet, 2 H, J ) 6.9), 2.43 (m, 12 H), 2.60 (m, 4 H),
3.00 (br s, 4 H), 3.45 (d, 2 H, J ) 13.5), 3.64 (d, 2 H, J ) 13.5),
3.79 (m, 2 H), 7.23 (m, 10 H); 13C NMR δ 2.85, 2.89, 10.64,
14.67, 23.91, 43.59, 51.53, 53.16, 54.47, 57.78, 58.46, 66.30,
66.39, 126.51, 127.74, 128.47, 135.25; HRMS, m/z calcd for
Spermidine/spermine N1-acetyltransferase activity was based
on quantitation of [14C]-N1-acetylspermidine formed by acetyl-
ation of SPD with [14C]-acetyl coenzyme A according to the
method of Libby et al.18 Cells treated with DENSPM were
positive controls.
Acu te a n d Ch r on ic Toxicity of P olya m in e An a logu es
in Mice. For acute toxicity assessments, the polyamine
analogues were administered to female CD-1 mice (Charles
River, Wilmington, MA) as a single ip injection.27 The animals
were scored 2 h after administration of drug; survivors were
observed for a period of 10 days after the treatment. On the
basis of the results from the acute toxicity trials, the chronic
toxicity dosing regimen was set up so that at least one test
group should have a high fraction of lethalities. In the chronic
toxicity regimen, mice were administered the polyamine
analogue as 1 ip dose/day for 5 days. Body weight and
observations of coat health and overall physical appearance
were recorded at the time of each injection. After the treatment
regimen had been completed, animals were monitored daily
for an additional 10 days.
C
1.18).
29H47N4O2 483.3699 (M + H), found 483.3702; [R]24 +57.6 (c
D
(2R,10S)-N1-Cyclop r op ylm et h yl-N11-et h yl-2,10-d ih y-
d r oxyn or sp er m in e Tetr a h yd r och lor id e (2). A solution of
20 (2.32 g, 4.81 mmol) in EtOH (200 mL) and 1 N HCl (38
mL) was degassed with N2, and 10% Pd-C (0.66 g) was
introduced. After the flask had been purged with N2 (three
times), the reaction mixture was stirred overnight under an
H2 atmosphere (1 atm). Solids were filtered through Celite,
and the filtrate was concentrated under reduced pressure. The
residue was recrystallized from aqueous EtOH to give 1.50 g
of 2 (70%) as white crystals: 1H NMR δ 0.39 (m, 2 H), 0.71
(m, 2 H), 1.11 (m, 1 H), 1.32 (t, 3 H, J ) 7.2), 2.20 (m, 2 H),
3.19 (m, 16 H), 4.33 (m, 2 H); 13C NMR δ 4.11, 7.05, 10.98,
23.01, 43.93, 45.38, 49.91, 49.99, 50.73, 50.79, 53.40, 63.52;
HRMS, m/z calcd for C15H35N4O2 303.2760 (M + H), found
Ack n ow led gm en t. We thank GelTex, Inc., Walth-
am, MA, for financial support. We appreciate the
technical assistance of Elizabeth M. Nelson and Dr.
Eileen Eiler-McManis’s assistance in the organization
and editing of the manuscript.
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-
Cell Cu ltu r e. Murine L1210 leukemia cells were main-
tained in logarithmic growth as a suspension culture in RPMI-
1640 medium (Gibco) containing 10% fetal bovine serum
(Sigma), 2% HEPES-MOPS buffer, and 1 mM aminoguani-
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growth after defined intervals of exposure.
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fractured in liquid nitrogen/hot water three times. Each
supernatant was frozen at -20 °C until analysis of polyamine
content by HPLC.26

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J M000532Q