Conformationally restricted analogues of 1N,12N-bisethylspermine: Synthesis and growth inhibitory effects on human tumor cell lines

Article abstract of DOI:10.1021/jm980172v

Eight analogues of ¹N, ¹²N-bisethylspermine (BES) with restricted conformations were synthesized in the search for new spermine mimetics with cytotoxic activities. By replacing the central butane segment of BES with a 1,2-disubstitut

Full text of DOI:10.1021/jm980172v

J . Med. Chem. 1998, 41, 4723-4732
4723
1
Con for m a tion a lly Restr icted An a logu es of N,12N-Biseth ylsp er m in e: Syn th esis
a n d Gr ow th In h ibitor y Effects on Hu m a n Tu m or Cell Lin es
Venodhar K. Reddy,† Aldonia Valasinas,† Aparajita Sarkar,† Hirak S. Basu,‡ Laurence J . Marton,§ and
Benjamin Frydman*,†
SLIL Biomedical Corp., 535 Science Drive, Suite C, Madison, Wisconsin 53711, and Departments of Human Oncology, of
Pathology and Laboratory Medicine, and of Oncology, McArdle Laboratory for Cancer Research, University of
Wisconsin-Madison, Madison, Wisconsin 53706
Received March 19, 1998
Eight analogues of 1N,12N-bisethylspermine (BES) with restricted conformations were synthe-
sized in the search for new spermine mimetics with cytotoxic activities. By replacing the central
butane segment of BES with a 1,2-disubstituted cyclopropane ring, a pair of cis/trans-isomers
was obtained that introduced a spatial constraint in the otherwise freely mobile butane chain.
An analogous pair of isomers was obtained when the butane segment was replaced with a
1,2-disubstituted cyclobutane ring or with a 2-butene residue. The six new BES analogues
thus obtained (three pairs of cis/trans-isomers) were growth inhibitory at low-micromolar
concentrations against four human tumor cell lines (A549, HT-29, U251MG, and DU145) but
were less growth inhibitory against two other human tumor cell lines (PC-3 and MCF7). 1N,12N-
Bisethylspermyne, where the central butane segment of BES was replaced by the rigid 2-butyne
segment, was devoid of growth inhibitory activity against five of the six human cell lines studied
(DU145 being the only exception), a clear indication of the importance of conformational mobility
4
at the N,9N-butane segment of BES for its biological activity. When the butane segment was
replaced by a benzene-1,2-dimethyl residue, the resulting BES analogue was devoid of growth
inhibitory activity despite its cisoid conformation. The cytotoxicity of the analogues does not
seem to be directly related to their uptake by the cells or to their effects on cellular polyamine
levels. BES analogues with restricted conformations but which contained the equivalent of a
two-carbon unit, rather than the natural four-carbon unit, at the central segment, such as
1,2-diaminocyclopropyl or 1,2-diaminocyclobutyl derivatives, were devoid of growth inhibitory
effects at the concentrations studied. The development of conformationally restricted polyamine
analogues appears to show promise in the further quest for polyamine-related therapeutic
agents with specificity of action.
In tr od u ction
Spermidine and spermine can cause DNA to condense
and aggregate and can induce both B-to-Z and B-to-A
transitions in certain DNA sequences.5 Molecular
mechanics studies of spermine-DNA interactions have
shown that, in a minimum energy conformation, sper-
mine is bound in a cisoidal conformation that wraps
around the major groove of the double helix.6 Spermine
and its RN,ωN-bisethylated higher and lower homo-
logues, as well as spermidine, have been shown to bind
to t-RNA.7 Using 1H NMR analysis spermine and
1N,12N-bisethylspermine (BES) were found to bind at
the TψC loop of t-RNAPhe. The binding is not of an
electrostatic nature, but rather a consequence of the
different hydrogen-bonding modes that can be estab-
lished between both types of molecules. Finally,
polyamines have recently been shown to bind to recep-
tors such as the N-methyl-D-glutamate (NMDA) recep-
tor and the glutamate receptor (Glu-R) and to block and
modulate a number of ion channels:8 results that open
new vistas in the pharmacology of the polyamines.
The polyamines (putrescine, spermidine, and sper-
mine) increase in proliferating tissues and are essential
for cellular growth and division.1,2 A number of
polyamine analogues show promise as anticancer agents.
They are able to kill cells and inhibit cell growth both
in vitro and in vivo.1 Most successful among these
analogues have been the RN,ωN-alkyl derivatives of
spermine’s higher and lower homologues, although
several alkylated diamines also show promise as inhibi-
tors of tumor cell proliferation.3 Of the many hypoth-
eses advanced to explain the biological effects of the
polyamines,1,2 the ones that explore their binding to
nucleic acids and to receptor targets are the most
compelling. Since spermidine and spermine are strong
bases, they are protonated at physiological pH1,2 and
can therefore bind to the negatively charged nucleic
acids either by electrostatic interactions or by hydrogen
bonding. Polyamines are known to interact with and
induce structural changes in DNA in cell-free systems.4
DNA-interacting drugs have long been of interest as
anticancer agents. Mechanical models of the DNA
double helix have created an image of a rigid structure;
however, experimental evidence suggests that DNA has
considerable flexibility.9 When designing polyamine
analogues that can bind to DNA, considered structural
* To whom correspondence should be addressed. Phone: 608-231-
3854. Fax: 608-231-3892. E-mail: bjf@mail.slil.wisc.edu.
† SLIL Biomedical Corp.
‡ Department of Human Oncology.
§ Departments of Pathology and Laboratory Medicine and of On-
cology.
10.1021/jm980172v CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/24/1998

4724 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Reddy et al.
Sch em e 1
1
F igu r e 1. Conformations of N,12N-bisethylspermine (BES)
and the trans/cis-isomer pair 5 and 9 (see text) resulting from
replacing the central butane segment in BES with trans- and
cis-1,2-dimethylcyclopropyl residues.
modifications include variations in the number and
distance of carbons between nitrogens and/or terminal
N-substitutions of different types.3 We report below the
design and synthesis of chiral analogues of spermine
that might bind DNA, t-RNA, or other polyamine
binding sites and due to their increased conformational
rigidity might do so in a modified and selective fashion.
The introduction of restriction in the free rotation about
the single bonds in a flexible molecule such as spermine
(which has myriad potential conformations) can con-
ceivably result in spatial rigidity that could introduce
bends, kinks, or loops at their binding domains. The
introduction of conformational restriction has been very
fruitful in the design of peptidomimetics,10 and a recent
report described the synthesis of chiral pyrrolidyl
polyamines.11 We followed the principle that in order
to be able to bind to biologically relevant sites, it would
be best to construct rigid analogues of spermine in which
the added atoms or bonds would affect the size and
molecular weight of the parent compound as little as
possible. The simple addition of a cyclopropyl ring to
the butane segment of spermine introduces chirality and
conformational restriction in an otherwise flexible mol-
ecule (Figure 1). The same is true for the introduction
of a cyclobutyl ring and of a double bond. The synthesis
Sch em e 2
Sch em e 3
R
of the N,ωN-bisethyl derivatives of the aforementioned
partially rigid spermine analogues is described below.
Ch em istr y
The synthesis of the cyclopropyl analogues of sper-
mine was achieved using the trans- and cis-1,2-bis-
(dihydroxymethyl)cyclopropanes as starting intermedi-
ates. Thus, the trans-isomer 1 was acylated with
mesitylenesulfonyl chloride to give 2 (Scheme 1). The
ester was then reacted with the sodium salt of N1-
ethylpropane-1,3-diamine dimesitylate (3). Displace-
ment of the mesitylenesulfonate residues on 2 gave the
tetramide 4. Deprotection of the amine groups using
established procedures3b,12 resulted in a 75% yield of the
tetrahydrochloride of 5. This approach was also found
to be useful in obtaining the cis-isomer 9. Starting with
the cis-bis(dihydroxymethyl) 6, esterification with mesi-
tylenesulfonyl chloride gave 7, reaction with the sodium
salt of 3 gave 8, and deprotection in acid medium gave
the tetrahydrochloride of 9. This sequence of reactions
also lent themselves to upscaling when larger amounts
of polyamines were needed.
The synthetic outline also allowed for the synthesis
of the cyclobutyl analogues (Scheme 2). The trans- and
cis-diols 12 and 16 were esterified to 13 and 17 and the
ester residues displaced with the sodium salt of 3 to give
14 and 18. Deprotection of the amino groups gave the
tetrahydrochlorides of 15 and 19.
The same outline led to the synthesis of the geo-
metrical isomers 23 and 27 (Scheme 3). Starting with
the trans- and cis-2-butenediols 20 and 24, by converting
them to the esters 21 and 25, and by reacting the latter

Conformationally Restricted Analogues of BES
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4725
Sch em e 4
Sch em e 6
Sch em e 5
with 3 as described above, it was possible to obtain 22
and 26. Deprotection of the amine groups yielded the
tetrahydrochlorides of 23 and 27 in good overall yields
(56% from the diol to the tetramine).
Of the synthetic approaches to the conformationally
restricted spermine analogues that were tried and
discarded, the use of the Mitsonobu reaction13 is worth
mentioning. The latter was used in an attempt to
convert the bis(hydroxymethyl) derivatives (e.g., 1, 6,
12, 16, 20, 24) into the corresponding bis(aminomethyl)
derivatives. As exemplified for 1 and 6 (Scheme 1), the
diols were brought into reaction with hydrazoic acid in
the presence of diisopropyl azodicarboxylate, followed
by triphenylphosphine. Finally the phosphoranes were
hydrolyzed with water, and the bases 10 and 11 were
isolated as their hydrochlorides. Yields never exceeded
20-35% with any of the diols. Moreover, in the case of
the diols 12 and 16, the reaction also led to the
formation of mixed 1-hydroxymethyl-2-aminomethyl
derivatives that further decreased the overall yield of
the reaction; hence, this approach was not pursued
further.
To further examine the influence of conformation on
the central butane segment, the synthesis of the bisethyl
derivative of spermyne (the triple-bond analogue of
bisethylspermine) 31 was carried out (Scheme 4). In
31 the four-carbon backbone is entirely rigid due to the
sp bond at the central carbons and allows no conforma-
tional isomerism. It was prepared by starting with the
commercially available butyne-1,4-diol (28) which was
mesitylated to 29, the latter brought into reaction with
3 to give the tetramide 30. Deprotection to 31 followed
the procedure mentioned above.
To further examine the influence of a bulky substitu-
ent on the butane segment of the analogues, the bisethyl
derivative 35 was prepared (Scheme 5). In 35 the
central four carbons are part of a 1,2-benzyldiamine
structure and are therefore restricted by the aromatic
sp2 bonds to a cisoid conformation analogous to that
present in 27 (Scheme 3). This analogue was con-
structed starting with commercially available benzene-
1,2-dimethanol (32) which was mesitylated to 33, the
latter brought into reaction with 3 to give 34. Depro-
tection to 35 followed the usual procedure.
cyclopropanes 36 and 43, it was possible to convert them
into the 1,2-diaminocyclopropanes 38 and 45. The
syntheses of the latter by other procedures have been
reported,14-16 but we found it more convenient to use
the above-mentioned method. The trans- and cis-1,2-
cyclobutane-diamines 49 and 53 are known.17 The
diamines were converted into the corresponding sulfo-
nylmesitylene derivatives 39, 46, 50, and 54, and the
latter were alkylated with N-ethyl-N-(3-bromopropyl)-
mesitylenesulfonamide (40) in the presence of sodium
hydride. The tetramides 41, 47, 51, and 55 thus
obtained were cleaved with 33% HBr in glacial acetic
acid in the presence of phenol, and the corresponding
tetramines 42, 48, 52, and 56 were isolated as their
tetrahydrochlorides.
Biologica l Resu lts a n d Discu ssion
Six human tumor cell lines were used to assay for the
growth inhibitory activities of the new polyamine ana-
logues (Table 1). As a reference, the growth inhibitory
activity of N1,N12-bisethylspermine (BES) (the flexible
chain polyamine analogue) was assayed on lung (A549),
colon (HT-29), prostate (PC-3 and DU145), breast
(MCF7), and brain (U251MG NCI) human tumor cells.
BES was effective against all cell lines (Table 1). The
When the isomeric cyclopropyl and cyclobutyl cis- and
trans-1,2-diamines were used as starting materials, they
were found to be easily alkylated to give the tetramines
(Scheme 6). By using the Curtius rearrangement on the
hydrazides of the trans- and cis-1,2-bis(ethoxycarbonyl)-

4726 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Reddy et al.
Ta ble 1. Inhibition of Cell Growth by Polyamine Analogues in
Ta ble 2. Polyamine and Analogue Levels in DU145 Cells
Treated with Polyamine Analoguesa
Human Tumor Cell Linesa
ID50 (µM)b
concentration (nmol/106 cells)
drug A549 HT-29
PC-3
MCF7 U251MG NCI DU145
concn
day of
drug
(µM) treatment
PU
SD
SP
analogue
BES
9
5
19
15
27
23
31
35
0.20
0.12
0.25
0.10
0.30
0.25
0.26
0.43
1.60
1.40
0.24
7.40 >31.25
12.40 >31.25
0.55
0.20
0.10
0.10
0.10
0.12
0.04
0.02
0.06
0.05
0.01
0.05
0.14
1.33
12.60
control
0
1
2
3
5
0.19
0.19
0.11
0.04
2.44 3.18
2.40 3.04
0.15 2.63
1.61 1.52
1.50 >31.25
25.50
1.60 >31.25 >31.25
1.60
1.40
3.60
1.40 >31.25
9.50
0.55
2.00
9
0.02
2.00
1
3
1
3
0.18
0.05
NQ
1.64 2.16
1.01 1.97
0.10 0.40
0.90
0.15
4.08
3.97
>31.25 >31.25 >31.25 >31.25
>31.25 >31.25 >31.25 >31.25
>31.25
>31.25
NQ
NQ
0.06
a
Exponentially growing cells were exposed to graded concentra-
tions of polyamine analogues for 6 days. The MTT assay was
performed to determine cell survival. ID50 values were determined
by extrapolation from a plot of relative changes in absorbance at
570 nm. Each value is an average of experiments performed in
quadruplicate. b ID50 represents the drug concentration that killed
50% of the cells.
5
0.02
2.00
1
3
1
3
0.20
0.08
0.55
0.05
1.92 2.48
1.41 2.73
2.63 2.64
1.38 2.28
0.14
0.06
0.33
0.18
15
27
31
0.02
5.00
3
5
3
5
0.162
0.081
0.036
0.031
2.13 3.54
1.6
0.15 0.49
0.06 0.20
0.07
0.05
1.75
1.84
2.10
conformationally restricted analogues 9, 5, 19, 15, 27,
and 23 were found to have good growth inhibitory effects
against A549, HT-29, U251MG NCI, and DU145 cells.
The ID50 values were of the same order of magnitude
as those found for BES (in the range from 0.01 to 2.00
µM). No significant differences in cytotoxicity were
observed between the trans- and cis-isomeric pairs (i.e.,
9 and 5, 19 and 15, 27 and 23).
0.02
5.00
3
5
3
5
0.11
0.04
0.06
0.03
1.79 2.99
1.73 1.71
0.02 0.09
0.02 0.07
0.04
0.03
2.76
2.54
0.
3
5
3
5
0.11
0.03
0.04
0.06
1.73 3.13
1.55 1.55
0.90 2.56
0.59 1.33
0.06
0.03
2.16
1.71
5.00
35
10.00
3
5
0.12
0.06
1.50 2.70
1.84 2.13
0.82
1.25
To check whether the cytotoxicities of the analogues
are due to their cellular uptake and/or effects on cellular
polyamine levels, we measured the cellular polyamine
and analogue concentrations in DU145 prostate tumor
cells treated with some of the polyamine analogues used
in our studies. The DU145 cells were chosen because
this cell line has shown good sensitivity to most
polyamine analogues used in our studies (Table 1). The
cellular polyamines and analogue concentrations are
listed in Table 2. With the exception of the trans-
cyclopropyl analogue 5, all other analogues are readily
taken up by the cells. At concentrations between 2 and
5 µM, the intracellular concentrations of analogues
(except 5) are comparable with the intracellular sper-
mine level in control untreated cells. While compounds
5, 9, 15, and 27 are highly cytotoxic to DU145 cells (ID50
e 0.06 µM), compounds 31 and 35 have very little
cytotoxic effect (ID50 > 1 µM). Thus, cytotoxicities of
the analogues do not seem to be directly related to
intracellular analogue concentrations. While com-
pounds 9, 15, and 27 at concentrations between 2 and
5 µM deplete cellular putrescine, spermidine, and sper-
mine levels within 5 days of treatment, compounds 5,
31, and 35 have no appreciable effect on cellular
polyamine levels.
With PC-3 and MCF7 cell lines the restricted ana-
logues did not exhibit strong growth inhibition (with the
exception of 23 in PC-3), at variance with the results
for BES. In PC-3 cells slightly different ID50 values
were observed for the trans-cyclopropyl analogue 5 and
for the cis-isomer 9. Both cyclobutyl isomers 15 and
19 were minimally growth inhibitory. The double bond
isomers 23 and 27 were active with slightly different
ID50 values. In MCF7 cells most of the isomers were
inactive; however, the cis-double-bond isomer 27 was
moderately active, while the trans-isomer 23 was inac-
tive. It is conceivable that the difference in growth
a
Each number is an average of two separate experiments. NQ,
not quantifiable; PU, putrescine; SD, spermidine; SP, spermine.
inhibition between the isomeric pairs may increase
when the analogues are assayed in vivo and additional
pharmacokinetic factors come into play.
The total lack of activity of bisethylspermyne (31) in
five cell lines (A549, HT-29, PC-3, MCF7, and U251MG)
is noteworthy. This is the rigid analogue of BES at its
central four-carbon segment, and its inability to adopt
any spatial compromise related to binding might relate
to its lack of growth inhibitory effects. Although 31
showed some growth inhibition in the DU145 prostate
cell line, its ID50 value was almost 100-fold higher than
that of several of the other analogues. These results
lend support to previous reports that suggested that the
hydrogens carried by the central nitrogen atoms may
be a major binding force in spermine-nucleic acid
interactions.6,7 The lack of growth inhibition found for
35 is also revealing. Although in 35 the N-ethylamino-
1N-propyl side chains are bound to the central four-
carbon chain by sp2 bonds, as is the case with 27 and
23, thus conferring to 35 a cisoid conformation, the
analogue carrying the aromatic ring is biologically
inactive. This again stresses the need not to depart too
greatly from the structures of the natural polyamines
when designing conformationally restricted analogues.
It is evident from the data shown in Table 2 that the
least active polyamines, i.e., 31 and 35, were taken up
by cells as efficiently as the most active ones. Thus,
cellular uptake does not seem to be a factor that
influences the cell growth inhibition found with the new
polyamine analogues. There appears to be a differential
effect in cell uptake related to cis/trans-isomerism: the
cis-isomer 9 entered the cells at much higher concentra-

Conformationally Restricted Analogues of BES
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4727
tr a n s-1,2-Bis((m esit ylen esu lfon yloxy)m et h yl)cyclo-
p r op a n e (2). Diol 120 (1.65 g, 16.18 mmol) was dissolved in
10 mL of pyridine, the solution cooled to 0 °C, and mesity-
lenesulfonyl chloride (9.13 g, 41.75 mmol) in 23 mL of pyridine
added slowly over a span of 15 min. The mixture was stirred
at 25 °C for 3 h and then poured onto ice (75 g), and the white
solid precipitate was filtered and recrystallized from ethanol:
tions than its trans-isomer 5. This uptake effect clearly
influenced the intracellular pools of the natural poly-
amines, which were more depleted in the presence of 9
than in the presence of 5. Cellular polyamine pools were
not depleted by the uptake of the weak cell growth
inhibitory 31 and 35. Thus, no clear-cut relationship
between the depletion of intracellular polyamine pools
and inhibition of cell growth can be drawn from the data
shown in Table 2, further evidence of the complexity of
the biological effects of polyamines (see Introduction).
1
2.8 g (37%); mp 81-82 °C; H NMR (CDCl3) δ 0.50-0.65 (m,
2H, cyPrH2), 1.10 (m, 2H, cyPrH), 2.31 (s, 3H, CH3), 2.34 (s,
3H, CH3), 2.60 (s, 6H, 2CH3), 2.61 (s, 6H, 2CH3), 3.80 (m, 4H,
2CH2), 6.97 (s, 2H, Ph), 6.99 (s, 2H, Ph); 13C NMR (CDCl3) δ
9.47, 16.36, 21.04, 22.57, 72.13, 130.62, 131.76, 139.79, 143.37;
MS-EI m/z 466 (M+), 452, 386, 118 (100%). Anal. Calcd for
C23H30O3S2: C, 66.03; H, 7.18. Found: C, 66.05; H, 7.15.
The tetramines 42, 48, 52, and 56 did not exhibit
growth inhibitory effects on the aforementioned cell
lines, as might be expected from polyamines having a
1,2-diaminoethane backbone.18
3,7,13,17-Tetr a k is(m esitylen esu lfon yl)-9,10-[(E)-1,2-cy-
clop r op yl]-3,7,13,17-tetr a a za octa d eca n e (4). Amide 3 (2.5
g, 5.37 mmol) was dissolved in 40 mL of anhydrous DMF, NaH
(95%, 300 mg) was slowly added under argon and the mixture
was stirred at 25 °C for 0.5 h, after which a solution of 2 (1.16
g, 2.49 mmol) in 35 mL of anhydrous DMF was added over a
period of 10 min. The stirred reaction mixture was heated to
70 °C for 4 h, then cooled to 0 °C, and quenched by the addition
of 10 mL of water. The mixture was extracted with ether (3
× 30 mL); the combined organic layers were washed with
water (4 × 30 mL) and then brine (2 × 25 mL), dried (Na2-
SO4), and evaporated to dryness. The oily residue was purified
by flash chromatography on silica gel using hexanes-ethyl
acetate (8:2) as eluant to give 4 as a low-melting white
semisolid (1.0 g, 40% yield): 1H NMR (CDCl3) δ 0.34 (m, 2H,
cyPrH2), 0.75 (m, 2H, cyPrH), 0.95 (t, J ) 7.1 Hz, 6H, 2CH3),
1.70 (m, 4H, 2CH2), 2.30 (s, 12H, 4CH3), 2.54 (s, 24H, 8CH3),
2.75 (m, 2H, NCH), 3.10 (m, 14H, NCH2), 6.90 (s, 4H, Ph),
6.93 (s, 4H, Ph); 13C NMR (CDCl3) δ 12.69, 15.69, 20.95, 22.75,
25.29, 40.17, 42.64, 43.40, 48.90, 131.92, 131.98, 132.99,
133.26, 140.08, 140.12, 142.34, 142.49; MS-FAB m/z 999.4 (M
+ 1)+, 815.3, 633.3, 533.2.
3,7,13,17-Tet r a a za -9,10-[(E)-1,2-cyclop r op yl]oct a d e-
ca n e (5) Tetr a h yd r och lor id e. Phenol (1.68 g, 17.8 mmol)
and hydrogen bromide (33%) in glacial acetic acid (8.9 mL)
were added in tandem to a solution of 4 (447 mg, 0.45 mmol)
in methylene chloride (5 mL) at 25 °C. The solution was
stirred for 48 h, after which water (6 mL) was added and the
aqueous layer was separated and extracted with methylene
chloride (3 × 8 mL). The aqueous layer was evaporated under
reduced pressure, the residue was taken up in 10 N sodium
hydroxide (3 mL), and the solution was extracted with
chloroform (12 × 6 mL). The pooled extracts were evaporated
to dryness, leaving behind a thick gum that was taken up in
dry ethyl ether. Hydrogen chloride was bubbled through the
solution to precipitate the tetrahydrochloride of 5 as a white
solid; 140 mg (75% yield). The product was recrystallized from
ethanol/ether: mp 270 °C dec; 1H NMR (D2O) δ 0.88 (dd, J )
6.8, 7.0 Hz, 2H, cyPrH2), 1.25 (t, J ) 6.1 Hz, 2H, cyPrH), 1.30
(t, J ) 7.3 Hz, 6H, 2CH3), 2.12 (pent, J ) 7.9 Hz, 4H, NCCH2),
2.89 (dd, J ) 8.5, 12.1 Hz, 2H, NCH2), 3.08-3.30 (m, 14H,
NCH2); 13C NMR (D2O) δ 13.62, 14.10, 17.25, 26.35, 46.70,
47.49, 47.81, 54.41; MS-ESI m/z 271.3 (M + 1)+, 190.1, 145.0
(100%). Anal. Calcd for C15H40Cl4N4: C, 43.06; H, 9.57; N,
13.40. Found: C, 42.99; H, 10.00; N, 13.35.
Con clu sion s
We report on the synthesis of conformationally re-
stricted analogues of 1N,12N-bisethylspermine (BES).
Restriction was achieved by introducing a cyclopropyl,
a cyclobutyl, or a double bond in the central butane
segment of BES. The cis/trans-isomeric pairs thus
obtained showed growth inhibition in several human
tumor cell lines. The difference between these isomer
pairs was not experimentally significant. This could be
due to an insufficient degree of restriction imposed on
the isomer pairs. Introduction of further restrictions
at other sites of the spermine chain might lead to more
pronounced differential effects among the isomers.
Nevertheless, the significant growth inhibition achieved
with several cell lines with these new synthetic poly-
amines opens a new vista in the development of
polyamine analogues with antiproliferative effects.
Exp er im en ta l Section
NMR spectra were obtained using a Bruker MSL 300
spectrometer. 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 (EI) were obtained on a Kratos MS-80RFA; mass
spectra (FAB) were obtained on a Autospec (VG) spectrometer
that was also used to secure ESI (electrospray) spectra. Matrix
assisted laser desorption ionization (MALDI) mass spectra
were obtained using a Bruker Reflex II spectrometer equipped
with a 337-nm laser and delayed extraction. MALDI and ESI
spectra of polyamines were secured by injecting their hydro-
chlorides.
N-Eth yl-N-(3-(m esitylen esu lfon yla m in o)p r op yl)m esit-
ylen esu lfon a m id e (3). 1N-Ethylpropane-1,3-diamine19 (5.8
g, 56.8 mmol) was dissolved in a mixture of 50 mL of 4 N
NaOH and 25 mL at dioxane, and a solution of mesitylene-
sulfonyl chloride (27.22 g, 124.9 mmol) in 100 mL of dioxane
was added dropwise to the stirred solution kept at 5 °C. Once
the addition was completed the solution was kept at 20 °C
during a further 18 h. It was then poured over an excess of
ice water; the precipitate was filtered, dried, and crystallized
from ethanol-water: 19.8 g (73%) of 3 obtained as a white
solid; mp 101-102 °C; 1H NMR (CDCl3) δ 0.95 (t, J ) 6.99
Hz, 3H, CH3), 1.68 (pent, J ) 6.25 Hz, 2H, NCCH2), 2.29 (s,
6H, 2CH3), 2.56 (s, 6H, 2CH3), 2.62 (s, 6H, 2CH3), 2.91 (m,
2H, NHCH2), 3.12 (q, J ) 7.0 Hz, 2H, NCH2), 3.31 (t, J ) 6.44
Hz, 2H, NCH2), 4.93 (br, NH), 6.93 (s, 2H, aromatic), 6.95 (s,
2H, aromatic); 13C NMR (CDCl3) δ 12.57, 20.93, 22.81, 22.89,
27.84, 39.26, 39.99, 42.29, 131.95, 132.05, 133.32, 138.90
140.00, 142.02, 142.57. Anal. Calcd for C23H34N2O4S2: C,
59.23; H, 7.30; N, 6.01. Found: C, 59.15; H, 7.26; N, 6.59.
ci s-1,2-B is ((m e s it y le n e s u lfo n y lo x y )m e t h y l)c y c lo -
p r op a n e (7) was obtained from 620 as described above for 2:
mp 90-91 °C (ethanol-water); 1H NMR (CDCl3) δ 0.35 (m,
1H, cyPrH2), 0.93 (m, 1H, cyPrH2), 1.32 (m, 2H, cyPrH), 2.32
(s, 6H, 2CH3), 2.60 (s, 12H, 4CH3), 3.01 (m, 4H, 2CH2), 6.99
(s, 4H, Ph); 13C NMR (CDCl3) δ 9.71, 15.25, 21.08, 22.61, 69.61,
130.63, 131.79, 139.82, 143.39; MS-EI m/z 466 (M+), 452, 266,
200, 185, 171, 119 (100%). Anal. Calcd for C23H30O3S2: C,
66.03; H, 7.18. Found: C, 66.01; H, 7.20.
3,7,13,17-Tetr a k is(m esitylen esu lfon yl)-9,10-[(Z)-1,2-cy-
clop r op yl]-3,7,13,17-tetr a a za octa d eca n e (8) was obtained
by the reaction of 7 with 3 as described for 4: mp 56-58 °C
(ethyl acetate-hexane) (64%); 1H NMR (CDCl3) δ 0.07 (m, 1H,
cyPrH2), 0.76 (m, 1H, cyPrH2), 0.95 (t, J ) 7.1 Hz, 8H, 2CH3
and cyPrH), 1.71 (m, 4H), 2.28 (s, 6H), 2.29 (s, 6H), 2.54 (s,

4728 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Reddy et al.
12H), 2.55 (s, 12H), 2.69-2.90 (m, 2H), 3.00-3.20 (m, 12H),
3.29-3.40 (m, 2H), 6.92 (s, 4H), 6.93 (s, 4H); 13C NMR (CDCl3)
δ 12.63, 13.92, 20.96, 22.73, 22.77, 25.50, 40.07, 42.65, 43.49,
45.17, 131.92, 133.00, 146.06, 142.35; MS-FAB, m/z 999.40
(M+), 815.30, 533.20, 167.10, 119.00 (100%). Anal. Calcd for
20.86, 22.65, 22.72, 22.89, 25.37, 33.95, 39.98, 42.60, 43.31,
45.46, 131.92, 133.17, 139.94, 142.25, 142.45; MS-FAB m/z
1012.4546.
3,7,13,17-Te t r a a za -9,10-[(Z)-1,2-cyclob u t yl]oct a d e -
ca n e (19) tetr a h yd r och lor id e was obtained (82%) from 18
following the procedure described for the synthesis of 15: mp
C
51H76N4O8S4: C, 61.20; N, 7.60; N, 5.60. Found: C, 61.18;
1
H, 7.62; N, 5.56.
271-272 °C dec; H NMR (D2O) δ 1.30 (t, 6H), 1.85 (m, 2H),
2.08-2.25 (m, 6H), 2.50 (br, 2H), 3.10-3.30 (m, 16H); 13C NMR
(D2O) δ 14.24, 26.44, 38.77, 46.80, 47.60, 48.41, 55.05; MS-
ESI m/z 286.31 (M+). Anal. Calcd for C16H42Cl4N4: C, 44.44;
H, 9.72; N, 12.96. Found: C, 44.39; H, 9.69; N, 12.90.
(E)-2-Bu t en e-1,4-d iyl Bis[m esit ylen esu lfon a t e] (21).
Diol 2022 (1.76 g, 20 mmol) and benzyltriethylammonium
bromide (270 mg, 1 mmol) were dissolved in 30 mL of a 50%
potassium hydroxide solution and 30 mL of dioxane. The
mixture was stirred at 5 °C, and mesitylenesulfonyl chloride
(8.72 g, 40 mmol) dissolved in 30 mL of dioxane was added
dropwise. When the addition was over, stirring was continued
for 2 h, water was then added, and the white precipitate was
filtered and crystallized from chloroform-hexane: 7.0 g (77%);
mp 119-120 °C; 1H NMR (CDCl3) δ 2.35 (s, 6H), 2.60 (s, 12H),
4.45 (d, 4H), 5.75 (br, 2H), 6.95 (s, 4H); 13C NMR (CDCl3) δ
20.96, 22.52, 67.96, 127.67, 131.69, 131.74, 139.79, 143.45; MS-
EI m/z 452 (M+), 253, 200, 183. Anal. Calcd for C22H28O6S2:
C, 58.40; H, 6.19. Found: C, 58.35; H, 6.22.
3,7,13,17-Tet r a a za -9,10-[(Z)-1,2-cyclop r op yl]oct a d e-
ca n e (9) tetr a h yd r och lor id e was prepared following the
procedure described for 5: mp 240 °C dec (75%); 1H NMR (D2O)
δ 0.43 (m, 1H), 1.0 (m, 1H), 1.29 (t, J ) 6.3 Hz, 8H), 2.04 (m,
4H), 2.97 (m, 8H), 3.11 (m, 8H); 13C NMR (D2O) δ 12.63, 13.53,
15.74, 26.89, 45.82, 47.30, 47.39, 50.58; MS-ESI m/z 271.3 (M
+ 1)+. Anal. Calcd for C15H80Cl4N4: C, 43.06; H, 9.57; N,
13.40. Found: C, 43.08; H, 10.01; N, 13.45.
tr a n s-1,2-Bis((m esit ylen esu lfon yloxy)m et h yl)cyclo-
bu ta n e (13) was obtained (83%) from the diol 1221 following
the procedure described for 2: mp 77-78 °C; 1H NMR (CDCl3)
δ 1.65 (m, 2H, cyBuH2), 1.95 (m, 2H, cyBuH2), 2.30 (s, 6H,
2CH3), 2.40 (m, 2H, 2cyBuH), 2.60 (s, 12H, 4CH3), 3.88 (d, 4H,
CH2O), 6.96 (s, 4H, Ph); 13C NMR (CDCl3) δ 20.67, 20.86, 22.39,
36.37, 71.28, 130.35, 131.59, 131.62, 139.60, 143.17; MS-EI m/z
480 (M+), 281, 183, 119 (100%). Anal. Calcd for C24H32O3S2:
C, 66.67; H, 7.41. Found: C, 66.63; H, 7.38.
3,7,13,17-Tet r a k is(m esit ylen esu lfon a m id o)-9,10-[(E)-
1,2-cyclobu tyl]-3,7,13,17-tetr a a za octa d eca n e (14). Amide
3 (4.6 g, 10 mmol) was dissolved in 25 mL of DMF, and 300
mg (10 mmol) of an 85% dispersion of sodium hydride in oil
was added while the solution was kept under N2 at 5 °C with
constant stirring. After 1 h a solution of 2.2 g (4.5 mmol) of
13 in 25 mL of DMF was slowly added, and the mixture was
kept at 75-80 °C for 4 h. The solution was then evaporated
to dryness in vacuo, the solid partitioned between chloroform
and water, and the organic layer separated, washed with
water, dried (Na2SO4), and evaporated to dryness. The residue
was finally purified by chromatography through a silica gel
column using hexane-ethyl acetate (8:2) as the eluant. Tet-
ramide 14 was recovered as a glassy oil: 3.0 g (66%); 1H NMR
(CDCl3) δ 0.93 (t, 6H, 2CH3), 1.30 (m, 2H, cyBuH2), 1.70 (m,
6H, cyBuH2, CH2), 2.10 (br, 2H, 2cyBuH), 2.28 (s, 12H, CH3),
2.52 (s, 24H, CH3), 2.85-3.20 (m, 14H, CH2), 3.30 (m, 2H,
-CHN-), 6.90, 6.92 (s, s, 8H, Ph); 13C NMR (CDCl3) δ 12.63,
20.93, 22.72, 22.81, 23.28, 24.97, 37.45, 39.95, 42.29, 43.68,
50.01, 131.91, 133.70, 139.97, 140.03, 142.31; MS-FAB m/z
1012.4546.
(E)-3,7,12,16-Tet r a k is(m esit ylen esu lfon yl)-3,7,12,16-
tetr a a za octa d ec-9-en e (22). This was obtained following the
procedure described for 14. Amide 3 (4.15 g, 9.2 mmol) was
transformed into its sodium salt by reaction with 276 mg (9.2
mmol) of NaH (85% dispersion in oil) and the latter brought
into reaction with 1.94 g (4.2 mmol) of diester 21. The residue
was obtained after evaporation of the chloroform solution and
crystallized from ethyl acetate-hexane: 3.9 g (92%); mp 145-
146 °C; 1H NMR (CDCl3) δ 0.95 (t, J ) 7.1 Hz, 6H, 2CH3),
1.65 (m, 4H, NCCH2), 2.29 (2, 12H, 4CH3), 2.53 (s, 12H, 4CH3),
2.55 (s, 12H, 4CH3), 2.99 (t, J ) 7.3 Hz, 8H, NCH2), 3.08 (q, J
) 7.1 Hz, 4H, NCH2), 3.65 (d, J ) 5 Hz, 4H, CdC-CH2), 5.45
(m, 2H, CHdCH), 6.92 (s, 4H, aromatic), 6.93 (s, 4H, aromatic);
13C NMR (CDCl3) δ 12.62, 20.90, 22.68, 22.77, 25.56, 40.02,
42.53, 43.79, 43.83, 128.51, 131.88, 132.00, 132.71, 133.00,
140.02, 140.17, 142.30, 142.66; MS-FAB m/z 985.4 (M + 1)+.
(E)-3,7,12,16-Tetr a a za octa d ec-9-en e (23) tetr a h yd r o-
ch lor id e was obtained (86%) from 22 following the procedure
1
used for 15: mp 303-304 °C dec; H NMR (D2O) δ 1.30 (t, J
) 7.4 Hz, 6H, 2CH3), 2.10 (m, 4H, NCCH2), 3.13 (m, 12H,
NCH2), 3.77 (m, 4H, NCH2), 6.07 (m, 2H, CHdCH); 13C NMR
(D2O) δ 13.34, 25.57, 45.90, 46.71, 46.80, 51.04, 131.25; MS-
MALDI m/z 257.2 (M + 1)+. Anal. Calcd for C15H40Cl4N4: C,
43.06; H, 9.57; N, 13.40. Found C, 43.09; H, 9.60; N, 13.48.
(Z)-2-Bu ten e-1,4-d iyl bis[m esitylen esu lfon a te] (25) was
obtained (75%) from 24 (Aldrich) by following the procedure
described for 21: mp 71-72 °C; 1H NMR (CDCl3) δ 2.25 (s,
3,7,13,17-Te t r a a za -9,10-[(E )-1,2-cyclob u t yl]oct a d e -
ca n e (15) Tetr a h yd r och lor id e. This was obtained following
the procedure described for 5. Tetramide 14 (1.0 g, 0.98 mmol)
was treated with phenol (2.6 g, 27.6 mmol) and 45 mL of 33%
hydrogen bromide. After the usual workup the tetrahydro-
chloride 15 (355 mg, 87%) was crystallized from aqueous
ethanol: mp 288-289 °C dec; 1H NMR (D2O) δ 1.30 (t, 6H,
CH3), 1.80 (m, 2H), 2.02-2.28 (m, 6H), 2.50 (br, 2H), 3.02-
3.30 (m, 16H); 13C NMR (D2O) δ 14.22, 26.43, 38.77, 46.82,
47.60, 48.40, 55.03; MS-ESI m/z 286.31 (M+). Anal. Calcd for
C16H42Cl4N4: C, 44.44; H, 9.72; N, 12.96. Found: C, 44.48;
H, 9.69; N, 12.93.
6H), 2.50 (s, 19H), 4.40 (d, 4H), 5.62 (br, 2H), 6.90 (s, 4H); 13
C
NMR (CDCl3) δ 20.90, 22.40, 63.66, 127.54, 131.71, 139.69,
143.46; MS-EI m/z 452 (M+), 253, 199, 183. Anal. Calcd for
C
22H28O6N2: C, 58.40; H, 6.19. Found: C, 58.45; H, 6.25.
(Z)-3,7,12,16-Tet r a k is(m esit ylen esu lfon yl)-3,7,12,16-
tetr a a za octod ec-9-en e (26) was prepared (88%) by conden-
sation of 25 with 3 following the procedure described for 22:
ci s-1,2-B is ((m e s it y le n e s u lfo n y lo x y )m e t h y l)c y c lo -
bu ta n e (17) was obtained (80%) from the diol 1621 following
the procedure described for 2: mp 92-93 °C; 1H NMR (CDCl3)
δ 1.72 (m, 2H), 2.05 (br, 2H), 2.31 (s, 6H), 2.57, 2.59 (s, 12H),
2.78 (br, 2H), 3.85-4.10 (m, 4H), 6.96 (s, 4H); 13C NMR (CDCl3)
δ 20.69, 20.89, 21.49, 22.41, 34.94, 69.14, 130.41, 131.66,
139.62, 139.65, 143.20, 143.25; MS-EI m/z 480 (M+), 281, 199,
183. Anal. Calcd for C24H32O3S2: C, 66.67; H, 7.41. Found:
C, 66.70; H, 7.49.
1
mp 143-144 °C; H NMR (CDCl3) δ 0.93 (t, J ) 7.1 Hz, 6H),
1.60-1.70 (m, 4H), 2.29 (s, 6H), 2.30 (s, 6H), 2.52 (s, 12H),
2.55 (s, 12H), 2.99 (m, 8H), 3.07 (q, J ) 7.1 Hz, 4H), 3.74 (d,
J ) 4.6 Hz), 6.90 (s, 4H, aromatic), 6.93 (s, 4H, aromatic); 13
C
NMR (CDCl3) δ 12.69, 20.93, 22.71, 22.80, 25.20, 40.12, 42.56,
43.25, 47.06, 129.50, 131.93, 132.03, 132.86, 133.25, 140.07,
140.12, 142.36, 142.62; MS-FAB m/z 985.4 (M + 1)+.
(Z)-3,7,12,16-Tetr a a za octa d ec-9-en e (27) tetr a h yd r o-
ch lor id e was obtained (87%) from 26 following the procedure
1
3,7,13,17-Tet r a k is(m esit ylen esu lfon a m id o)-9,10-[(Z)-
1,2-cyclobu tyl]-3,7,13,17-tetr a a za octa d eca n e (18) was ob-
tained (70%) as an oil from the condensation of 17 with 3
following the procedure described for 14: 1H NMR (CDCl3) δ
0.95 (t, 6H), 1.30 (m, 4H), 1.70 (m, 6H), 2.30 (s, 12H), 2.58 (s,
24H), 3.10 (m, 16H), 6.95 (s, 8H); 13C NMR (CDCl3) δ 12.53,
used for 5: mp above 300 °C dec; H NMR (D2O) δ 1.30 (t, J
) 7.3 Hz, 6H), 2.10-2.59 (m, 4H), 3.05-3.25 (m, 12H), 3.87
(d, J ) 4.8 Hz, 4H), 5.98 (t, J ) 4.8 Hz, 2H); 13C NMR (D2O)
δ 13.35, 25.69, 45.93, 46.70, 46.96, 47.02, 129.31; MS-MALDI
m/z 257.1 (M + 1)+. Anal. Calcd for C15H40Cl4N4: C, 43.06;
H, 9.57; N, 13.40. Found: C, 43.02; H, 9.51; N, 13.45.

Conformationally Restricted Analogues of BES
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4729
2-Bu tyn e-1,4-d iyl bis[m esitylen esu lfon a te] (29) was
obtained (80%) from 28 (Aldrich) by following the procedure
described for 21: mp 105-106 °C (ethyl acetate-hexane); 1H
NMR (CDCl3) δ 2.31 (s, 6H), 2.60 (s, 12H), 4.52 (s, 4H), 6.98
(s, 4H); 13C NMR CDCl3 δ 20.93, 22.48, 56.13, 80.41, 130.65,
131.67, 139.98, 143.67; MS-EI m/z 450 (M+). Anal. Calcd for
C22H26O6S2: C, 58.67; H, 5.78. Found: C, 58.64; H, 5.75.
3,7,12,16-Tet r a k is(m esit ylen esu lfon yl)-3,7,12,16-t et -
r a a za octa d ec-9-yn e (30) was prepared (61%) by condensa-
tion of 29 with 3 following the procedure described for 22: mp
described for the synthesis of 10: mp 230 °C dec; 1H NMR
(D2O) δ 0.71-0.87 (m, 2H), 1.10-1.29 (m, 2H), 2.85 (m, 2H),
3.09 (m, 2H); 13C NMR (D2O) δ 13.19, 18.55, 46.53. Anal. Calcd
for C5H10Cl2N2: C, 35.50; H, 5.92; N, 16.57. Found: C, 35.47;
H, 5.89; N, 16.54.
tr a n s-1,2-Cyclopr opan edicar boxydih ydr azide (37). Hy-
drazine monohydrate (3.38 mL, 80 mmol) was added to a
solution of diethyl ester 3620 (3.5 mL, 20 mmol) in ethanol (10
mL), and the mixture was heated to reflux overnight. The
reaction was cooled and diluted with chloroform (20 mL); the
precipitate was filtered, dried, and recrystallized from etha-
nol: 2.53 g (80%); mp 227 °C (lit.23 mp 228 °C); 1H NMR (D2O)
δ 1.35 (dd, J ) 7.8 Hz, 2H, cyPrCH), 2.04 (dd, J ) 7.8 Hz, 1H,
cyPrCH); 13C NMR (D2O) δ 15.5, 24.0, 176.
tr a n s-1,2-Cyclop r op a n ed ia m in e (38) Dih yd r och lor id e.
Hydrazide 37 (1.58 g, 10 mmol) dissolved in a mixture of 4.5
mL of concentrated HCl and 9 g of ice was topped with 10 mL
of ethyl ether, and a solution of sodium nitrite (1.73 g, 25
mmol) in 4 mL of water was slowly added at 10 °C. The
organic layer was then separated, the aqueous layer was
extracted with ether (3 × 20 mL), and the ether extracts were
pooled, dried (CaCl2), and diluted with anhydrous toluene (30
mL). The ethyl ether was distilled off using a fractionation
column condenser, and the remaining toluene solution was
heated to 85 °C until the evolution of nitrogen ceased and then
for an additional 10 min. While still hot, the solution was
poured into preheated (60 °C) concentrated HCl (8 mL).
Toluene was distilled off under vacuum, and anhydrous
ethanol (15 mL) was added to the flask and then distilled off.
This process was repeated twice to obtain a cream-colored solid
that was digested with cold ethanol and suction-filtered to
afford 0.645 g (45%) of 38: mp 220 °C dec (from ethanol) (lit.14
mp 210 °C); 1H NMR (D2O) δ 1.48 (dd, 2H), 3.13 (dd, 2H). Anal.
Calcd for C3H10Cl2N2: C, 24.82; H, 6.89; N, 19.31. Found: C,
24.79; H, 6.85; N, 19.28.
1
165-166 °C (ethyl acetate-hexane); H NMR (CDCl3) δ 1.08
(t, 6H), 1.75 (m, 4H), 2.28 (s, 12H), 2.55 (brs, 24H), 3.10 (m,
12H), 3.98 (s, 4H), 6.95 (m, 8H); 13C NMR (CDCl3) δ 12.70,
20.86, 22.64, 25.14, 34.85, 40.22, 42.62, 43.37, 78.80, 131.99,
132.26, 133.21, 140.26, 142.28, 142.71; MS-FAB m/z 982 (M+).
Anal. Calcd for C50H70N4O8S4: C, 61.10; H, 7.13; N, 9.33.
Found: C, 61.08; H, 7.15; N, 9.38.
3,7,12,16-Tetr a a za octa d ec-9-yn e (31) tetr a h yd r och lo-
r id e was obtained (86%) from 30 following the procedure
described for 5:23 mp dec above 250 °C; 1H NMR (D2O) δ 1.29
(t, 6H), 2.13 (m, 4H), 3.14 (m, 12H), 4.06 (s, 4H); 13C NMR
(D2O) δ 13.34, 25.52, 39.45, 45.90, 45.64, 46.71, 81.32; MS-
MALDI m/z 255 (M + 1)+. Anal. Calcd for C14H34N4Cl4: C,
42.00; H, 8.50; N, 14.00. Found: C, 42.15; H, 8.68; N, 14.25.
1,2-Dim eth ylben zen e 1,2-bis(m esitylen esu lfon a te) (33)
was obtained (66%) from 32 (Aldrich) by following the proce-
dure described for 21: mp 128-129 °C (ethyl acetate-hexane);
1H NMR (CDCl3) δ 2.31 (s, 6H), 2.57 (s, 12H), 5.00 (s, 4H),
6.95 (s, 4H), 7.26 (m, 4H); 13C NMR (CDCl3) δ 20.97, 22.49,
67.78, 129.49, 130.19, 131.74, 132.39, 138.82, 143.43; MS-FAB
m/z 502 (M+). Anal. Calcd for C26H30O6S2: C, 62.15; H, 5.98.
Found: C, 62.11; H, 5.94.
9,10-Ben zo-3,7,12,16-tetr a k is(m esitylen esu lfon yl)-3,7,-
12,16-tetr a a za octod eca n e (34) was prepared (72%) by con-
densation of 33 with 3 following the procedure described for
22: mp 120-121 °C (ethyl acetate-hexane); 1H NMR (CDCl3)
δ 0.92 (t, 6H), 1.45 (m, 4H), 2.32, 2.38 (s, 12H), 2.48 (s, 12H),
2.65 (s, 12H), 2.75 (m, 4H), 2.85 (t, 4H), 2.95 (q, 4H), 4.30 (s,
4H), 6.88 (s, 4H), 7.02 (s, 4H), 7.28 (s, 4H); MS-FAB m/z 1034
(M+), 851. Anal. Calcd for C54H74N4O8S4: C, 62.67; H, 7.16;
N, 5.42. Found: C, 62.63; H, 7.20; N, 5.40.
t r a n s-1,2-C y c lo p r o p a n e d iy lb is (m e s it y le n e s u lfo n -
a m id e) (39). Dihydrochloride 38 (145 mg, 1 mmol) was
dissolved in 4 mL of dioxane/water (1:1), while maintaining a
pH of ca. 11 by the addition of 5% potassium hydroxide.
A
solution of mesitylenesulfonyl chloride (875 mg, 4 mmol) in 5
mL of dioxane was then slowly added, the upper layer of the
mixture was decanted, and the gummy residue was triturated
with hexane to afford 330 mg (76%) of 39 as a white solid that
was recrystallized from chloroform/hexane: mp 189-191 °C;
1H NMR (CDCl3) δ 0.9 (t, J ) 8.0 Hz, 2H, cyPrCH2), 2.24 (t, J
) 8 Hz, 2H, cyPrCH), 2.30 (s, 6H, 2CH3), 2.55 (s, 12H, 4CH3),
5.00 (s, 2H, NH), 6.75 (s, 4H, aromatic); 13C NMR (CDCl3) δ
14.16, 20.98, 22.92, 30.92, 132.11, 132.89, 139.20, 142.79; MS-
EI m/z 408 (M+). Anal. Calcd for C21H28N2S2O4: C, 61.76; H,
6.86; N, 6.86. Found: C, 61.70; H, 6.91; N, 6.90.
9,10-Ben zo-3,7,12,16-tetr a a za octa d eca n e (35) tetr a h y-
d r och lor id e was obtained (72%) from 35 following the
procedure described for 5: mp dec above 260 °C; 1H NMR
(D2O) δ 1.31 (t, 6H), 2.18 (m, 4H), 3.18 (m, 8H), 3.27 (m, 4H),
4.46 (s, 4H), 7.62 (s, 4H); 13C NMR (D2O) δ 13.31, 13.40, 25.71,
45.90, 46.75, 47.39, 50.91, 133.16, 133.38, 134.36; MS-MALDI
m/z 307 (M + 1)+. Anal. Calcd for C18H38N4Cl4: C, 47.79; H,
8.41; N, 12.39. Found: C, 47.73; H, 8.38; N, 12.19.
tr a n s-1,2-Bis(a m in om eth yl)cyclop r op a n e (10) Dih y-
d r och lor id e. A 0.4 M solution of hydrazoic acid in toluene
(102 mL) was added to a solution of diol 1 (1.7 g, 17.01 mmol)
in anhydrous THF (10 mL). This was followed by the addition
of a solution of diisopropyl azidodicarboxylate (7.48 g, 36.99
mmol) in THF (15 mL) and then by a solution of triphen-
ylphosphine (21.6 g, 92.35 mmol) in THF (30 mL). The
temperature of the mixture was kept at 25 °C by regulating
the rate of addition of the latter solution. Stirring was
maintained for 1 h at 25 °C and then for 18 h at 50 °C. Water
(3.5 mL) was then added, the mixture was stirred at 50 °C for
a further 6 h, the solvent was then removed under vacuum,
and the residue was partitioned between methylene chloride
(100 mL) and 1 N HCl (100 mL). The aqueous phase was
separated and repeatedly washed with methylene chloride (4
× 100 mL). Evaporation of the aqueous solution under
reduced pressure (40 °C) left behind 10 as a white solid that
was recrystallized from methanol/ether (0.650 g, 25%): mp 230
3,7,10,14-Tetr a k is(m esitylen esu lfon yl)-8,9-[(E)-1,2-cy-
clop r op yl]-3,7,10,14-t et r a a za h exa d eca n e (41). Sodium
hydride (0.111 g, 4.4 mmol) was added to a solution of 39 (0.872
g, 2 mmol) in anhydrous DMF (40 mL) at 0 °C, the mixture
was stirred for 30 min, a solution of 403b (1.531 g, 4.4 mmol)
in anhydrous DMF (50 mL) was then slowly added, and the
mixture was further stirred at 25 °C for 18 h. The reaction
was quenched with water (8 mL) and extracted with ether (3
× 25 mL); the combined organic layers were washed with
water (4 × 30 mL) and then with brine (2 × 25 mL), dried
(Na2SO4), and evaporated to dryness. The residue was filtered
through a silica gel column using 8:1 hexane-ethyl acetate
as the eluant: 1.7 g (77%) of 41 recovered from the eluates;
1
mp 60-62 °C; H NMR (CDCl3) δ 0.57 (dd, J ) 6 and 8 Hz,
2H, cyPrCH2), 0.99 (t, J ) 8 Hz, 6H, 2CH3), 1.7-1.9 (m, 4H,
NCH2CH2), 2.25 (s, 6H, 2CH3), 2.29 (s, 6H, 2CH3), 2.50 (s, 12H,
4CH3), 2.55 (s, 12H, 4CH3), 2.56-2.67 (m, 2H, cyPrCH), 2.87-
3.19 (m, 12H, 6NCH2), 6.86 (s, 4H, aromatic), 6.90 (s, 4H,
aromatic); MS-FAB m/z 971.4 (M + 1)+, 787, 605, 295, 119
(100%). Anal. Calcd for C49H70N4S4O8: C, 60.61; H, 7.22; N,
5.77. Found: C, 60.59; H, 7.18; N, 5.70.
1
°C dec; H NMR (D2O) δ 0.40-0.53 (m, 1H, cyPrCH), 1.00-
1.15 (m, 1H, cyPrCHH), 1.30-1.48 (m, 2H, cyPrCH), 2.75-
2.95 (m, 2H, NCH2), 3.20-3.35 (m, 2H, NCH2). Anal. Calcd
for C5H10Cl2N2: C, 35.50; H, 5.92; N, 16.57. Found: C, 35.56;
H, 6.01; N, 16.60.
3,7,10,14-Te t r a a za -8,9-[(E )-1,2-cyclop r op yl]h e xa d e -
ca n e (42) tetr a h yd r och lor id e was obtained (77%) from 41
following the procedure described for 5: mp 240 °C dec (from
cis-1,2-Bis(a m in om eth yl)cyclop r op a n e (11) d ih yd r o-
ch lor id e was obtained (35%) from 6 following the procedure

4730 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Reddy et al.
ethanol); 1H NMR (D2O-acetone-d6) δ 1.23 (t, J ) 6.8, 2H,
cyPrCH2), 1.31 (t, J ) 7.3 Hz, 6H, 3CH3), 2.00-2.10 (m, 4H,
NCCH2), 2.81 (t, J ) 6.6 Hz, 2H cyPrCH), 3.00-3.20 (m, 12H);
13C NMR (D2O-acetone-d6) δ 13.33, 13.73, 26.64, 37.63, 45.76,
47.27, 47.83; MS-ESI m/z 243.4 (M + 1)+, 163.3, 141.3. Anal.
Calcd for C13H34Cl4N4: C, 40.21; H, 8.76; N, 14.43. Found:
C, 40.27; H, 8.80; N, 14.49.
3,7,10,14-T e t r a a za -8,9-[(E )-1,2-c y c lo b u t y l]h e x a d e -
ca n e (52) tetr a h yd r och lor id e was obtained (40%) from 51
following the procedure described for 5: mp 275 °C dec; 1H
NMR (D2O) δ 1.20 (t, 6H), 1.80-2.21 (m, 6H), 2.35 (m, 2H),
3.08 (m, 12H), 4.00 (m, 2H); 13C NMR (D2O) δ 14.20, 22.57,
26.34, 46.26, 46.76, 47.44, 57.57; MS-ESI m/z 257.2 (M + 1)+,
172.2. Anal. Calcd for C14H36Cl4N4: C, 65.62; H, 8.95; N,
13.93. Found: C, 65.68; H, 9.00; N, 13.90.
cis-1,2-Cyclop r op a n ed ica r boxyd ih yd r a zid e (44) was
obtained (87%) from 4320 following the procedure described for
3,7,10,14-Tetr a k is(m esitylen esu lfon yl)-8,9-[(Z)-1,2-cy-
clobu tyl]-3,7,10,14-tetr a a za h exa d eca n e (55) was prepared
in 50% yield from 54 following the procedure described for
1
37: mp 196-198 °C (lit.24 mp 196 °C); H NMR (D2O) δ 1.25
(m, 1H, cyPrCH2), 1.45 (ddd, J ) 5.0, 6.0, 5.5 Hz, 1H, cyPrCH2),
2.05 (dd, J ) 6.0, 9.0 Hz, 2H, cyPrCH); 13C NMR (D2O) δ 12.97,
23.88, 175.0.
1
41: glassy oil; H NMR (CDCl3) δ 0.98 (t, 6H), 1.50 (m, 4H),
1.80 (br, 2H), 2.12 (br, 2H), 2.30 (s, 6H), 2.52, 2.54 (s, 12H),
2.82 (m, 4H), 2.93-3.23 (m, 8H), 4.20 (br, 2H), 6.93 (s, 8H);
13C NMR (CDCl3) δ 12.69, 20.94, 22.75, 22.82, 24.63, 25.94,
40.14, 42.16, 43.25, 55.43, 131.89, 132.23, 133.51, 135.08,
139.52, 140.16, 142.22, 142.88; MS-FAB m/z 985.4 (M + 1)+,
801.3, 619.3.
cis-1,2-Cyclop r op a n ed ia m in e (45) d ih yd r och lor id e was
obtained (58%) from 44 following the procedure described for
1
38: mp 220 °C (lit.15 mp 220 °C); H NMR (D2O) δ 1.25 (m,
2H, cyPrCH2), 2.9 (dd, 2H, J ) 6.0, 8.0 Hz, cyPrCH). Anal.
Calcd for C3H10Cl2N2: C, 24.82; H, 6.89; N, 19.31. Found: C,
24.89; H, 7.02; N, 19.50.
3,7,10,14-T e t r a a za -8,9-[(Z)-1,2-c y c lo b u t y l]h e x a d e -
ca n e (56) tetr a h yd r och lor id e was obtained from 55 in 37%
yield following the procedure described for 5: mp dec above
270 °C; 1H NMR (D2O) δ 2.10-2.25 (m, 4H), 2.35-2.55 (m,
4H), 3.08-3.30 (m, 12H), 4.15 (m, 2H); 13C NMR (D2O) δ 14.12,
25.04, 26.28, 46.72, 47.31, 47.46, 57.98; MS-ESI m/z 257.3 (M
+ 1)+, 172.3. Anal. Calcd for C14H36Cl4N4: C, 65.62; H, 8.95;
N, 13.93. Found: C, 65.70; H, 8.99; N, 13.97.
cis-1,2-Cyclop r op a n ed iylbis(m esitylen esu lfon a m id e)
(46) was obtained (95%) from 45 following the procedure
described for 39: mp 175-177 °C; 1H NMR (CDCl3) δ 0.80 (m,
1H), 0.95 (m, 1H), 2.25 (m, 2H), 2.34 (s, 6H), 2.65 (s, 12H),
5.25 (br, 2H), 7.00 (s, 4H, aromatic); 13C NMR (CDCl3) δ 13.5,
20.96, 22.96, 28.00, 132.08, 133.00, 139.45, 142.09. Anal.
Calcd for C21H28N2O4S2: C, 61.76; N, 6.86; N, 6.86. Found:
C, 61.80; H, 6.83; N, 6.92.
Cell Cu ltu r e. The human lung adenocarcinoma cell line,
A549, was a gift from Dr. M. Eileen Dolan, University of
Chicago, Department of Medicine, and was grown in Ham’s
F-12K medium (Fisher Scientific, Itasca, IL) supplemented
with 10% fetal bovine serum and 2 mM l-glutamine. The
human colon carcinoma cell line, HT-29, and the human breast
carcinoma cell line, MCF7, were obtained from the American
Type Culture Collection, Rockville, MD. HT-29 cells were
grown in McCoy’s 5A medium (Gibco, BRL, Gaithersburg, MD)
supplemented with 10% fetal bovine serum. MCF7 cells were
grown in Richter’s improved modified Eagle’s medium supple-
mented with 10% fetal bovine serum and 2.2 g/L sodium
bicarbonate. The human prostate adenocarcinoma cell lines,
PC-3 and DU145, were gifts from Dr. George Wilding, Uni-
versity of Wisconsin Comprehensive Cancer Center and the
Department of Medicine, and were grown in Dulbecco’s modi-
fied Eagle’s medium supplemented with 5% fetal bovine serum.
The malignant glioma cell line, U251MG NCI, was obtained
from the brain tumor tissue bank at the University of
California, San Francisco Department of Neurosurgery, and
was grown in Dulbecco’s modified Eagle’s medium supple-
mented wth 10% fetal bovine serum. Both A549 and MCF7
cells were grown in 100 units/mL penicillin and 100 µg/mL
streptomycin. HT-29 and U251MG NCI cells were grown in
50 µg/mL gentamycin. PC-3 and DU145 cells were maintained
in 1% antibiotic antimycotic solution (Sigma, St. Louis, MO).
All cell cultures were maintained at 37 °C in 5% CO2/95%
humidified air.
MTT Assa y.25 Exponentially growing monolayer cells were
plated in 96-well plates at a density of 500 cells/well and
allowed to grow for 24 h. Serially diluted drug solutions were
added such that the final drug concentrations in the treatment
media were between 0 and 35 µM. Six days after drug
treatment, 25 µL of a Dulbecco’s phosphate-buffered saline
solution containing 5 mg/mL MTT (thiazolyl blue) (Sigma) was
added to each well and incubated for 4 h at 37 °C. Then 100
µL of lysis buffer (20% sodium dodecyl sulfate, 50% N,N-
dimethylformamide, and 0.8% acetic acid, pH 4.7) was added
to each well and incubated for an additional 22 h. A microplate
reader (E max, Molecular Devices, Sunnyvale, CA) set at 570
nm was used to determine the optical density. Results were
plotted as a ratio of the optical density in drug-treated wells
to the optical density in wells treated with vehicle alone. The
ID50 values were determined by fitting the plot of optical
density vs concentration of analogues to a sigmoidal function
using Softmax Pro 2.0 4-parameter curve fitting software that
was developed based on Marquadt-Levenberg algorithm
(Molecular Devices, Sunnyvale, CA).
3,7,10,14-Tetr a k is(m esitylen esu lfon yl)-8,9-[(Z)-1,2-cy-
clop r op yl]-3,7,10,14-t et r a a za h exa d eca n e (47) was ob-
tained (30%) from 46 following the procedure described for
41: mp 74-75 °C; 1H NMR (CDCl3) δ 0.31 (m, 1H), 0.99 (t, J
) 8 Hz, 6H), 1.06 (m, 1H), 1.60 (m, 4H), 2.30 (s, 12H), 2.55 (s,
12H), 2.58 (s, 12H), 2.63 (m, 2H), 3.10 (m, 12H, 6NCH2), 6.90
(s, 4H, aromatic), 6.93 (s, 4H, aromatic); MS-FAB m/z 971.4
(M + 1)+, 787, 605, 154 (100%), 199. Anal. Calcd for
C49H70N4S4O8: C, 60.61; H, 7.22; N, 5.77. Found: C, 60.68;
H, 7.28; N, 6.00.
3,7,10,14-T e t r a za -8,9-[(Z)-1,2-c y c lo p r o p y l]h e xa d e -
ca n e (48) tetr a h yd r och lor id e was obtained (48%) from 47
1
following the procedure described for 42: mp 240 °C dec; H
NMR (D2O-acetone-d6) δ 0.75 (m, 1H), 1.16 (ddd, J ) 7.2, 8.1,
7.2 Hz, 1H), 1.29 (t, J ) 7.4 Hz, 6H), 2.04 (pent, J ) 7.5 Hz,
4H), 2.69 (dd, J ) 6.8, 7.4 Hz, 2H), 2.98-3.24 (m, 12H); 13C
NMR (D2O-acetone-d6) δ 12.44, 13.43, 27.12, 35.86, 45.89,
47.48, 48.06; MS-ESI m/z 243.3 (M + 1)+ (100%), 163, 142.
Anal. Calcd for C13H34Cl4N4: C, 40.21; H, 8.76; N, 14.43.
Found: C, 40.01; H, 8.69; N, 13.39.
t r a n s-1,2-C y c lo b u t a n e d i y lb i s (m e s i t y le n e s u lfo n -
a m id e) (50) was obtained (65% yield) from 4917 following the
procedure described for 39: mp 201-202 °C (methanol-
water); 1H NMR (CDCl3) δ 1.35 (m, 2H), 1.95 (m, 2H), 2.30 (s,
6H), 2.60 (s, 6H), 2.60 (s, 12H), 3.32 (m, 2H), 5.18 (brd, 2H),
6.95 (s, 4H); 13C NMR (CDCl3) δ 20.96, 22.88, 23.80, 54.68,
132.09, 134.10, 139.16, 142.80; MS-EI m/z 451 (M + 1)+, 267,
183. Anal. Calcd for C22H30N2O4S2: C, 58.67; H, 6.67; N, 6.22.
Found: C, 58.61; H, 6.71; N, 6.27.
cis-1,2-Cyclobu tan ediylbis(m esitylen esu lfon am ide) (54)
was obtained (70%) from 5317 following the procedure described
1
for 50: mp 192-193 °C (methanol-water); H NMR (CDCl3)
δ 1.90-2.18 (m, 4H), 2.35 (s, 6H), 2.60 (s, 12H), 3.63 (br, 2H),
5.41 (br, 2H), 6.96 (s, 4H); 13C NMR (CDCl3) δ 20.95, 22.89,
25.32, 51.20, 132.04, 134.08, 139.30, 142.51; MS-EI m/z 451
(M + 1)+, 267, 183. Anal. Calcd for C22H30N2O4S2: C, 58.67;
H, 6.67; N, 6.22. Found: C, 58.72; H, 6.70; N, 6.00.
3,7,10,14-Tetr a k is(m esitylen esu lfon yl)-8,9-[(E)-1,2-cy-
clobu tyl]-3,7,10,14-tetr a a za h exa d eca n e (51) was prepared
(60%) from 50 following the procedure described for 41:
1
colorless, glassy oil; H NMR (CDCl3) δ 0.98 (t, 6H), 1.55 (br,
6H), 1.82 (br, 2H), 2.28, 2.30 (s, 12H), 2.56-2.58 (s, 24H), 3.00
(m, 12H), 4.20 (br, 2H), 6.93-6.96 (s, 8H); 13C NMR (CDCl3) δ
12.70, 20.94, 21.70, 22.76, 22.90, 28.20, 40.20, 41.54, 43.16,
55.48, 131.91, 132.10, 133.02, 133.18, 140.23, 142.80, 143.17;
MS-FAB m/z 985.4 (M + 1)+, 801.4, 619.3.

Conformationally Restricted Analogues of BES
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4731
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P olya m in e An a lysis. Cells were incubated with the
polyamine analogues for the appropriate number of days (see
Biological Results and Discussion). Approximately 0.5-1.0 ×
106 cells were harvested by trypsinization and washed twice
with cold (4 °C) isotonic phosphate buffer (pH 7.4). Cells were
sonicated in 100-200 µL of 0.6 N perchloric acid and centri-
fuged at 8000g for 5 min, and the supernatant was carefully
separated for analysis. Between 50 and 100 µL of the
supernatant was fluorescence-labeled by derivatizing with
dansyl chloride. Labeled polyamines were loaded onto a C-18
high-performance liquid chromatography column and sepa-
rated by gradient elution with acetonitrile/water at 50 °C
following a published procedure.26 Peaks were detected and
quantitated using a Perkin-Elmer HPLC fluorescence monitor
coupled with a Spectra Physics peak integrator. Because
polyamine levels vary with environmental conditions, control
cultures were sampled for each experiment.
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Ack n ow led gm en t. We are grateful to Mr. David
Snyder (University of Wisconsin-Madison) for help in
securing the EI and FAB mass spectra and to Ms.
Martha M. Vestling (University of Wisconsin-Madison)
for securing the MALDI spectra. Our gratitude also goes
to Ms. Kendra Tutsch (University of Wisconsin-
Madison) for performing the HPLC analyses.
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