Synthesis and evaluation of hydroxylated polyamine analogues as antiproliferatives

Article abstract of DOI:10.1021/jm990375z

The synthesis of four hydroxylated polyamine analogues, (2R,10R)- N¹,N¹¹-diethyl-2,10-dihydroxynorspermine, (2S,10S)-N¹,N¹¹-diethyl-2,10- dihydroxynorspermine, (3S,12S)-N¹,N¹⁴-diethyl-3,12-

Full text of DOI:10.1021/jm990375z

224
J . Med. Chem. 2000, 43, 224-235
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, J o¨rg Bussenius,§ J ames S. McManis, Ronald L. Merriman,‡
Richard E. Smith, Hua Yao, and William R. Weimar
Department of Medicinal Chemistry, University of Florida, J . Hillis Miller Health Science Center, Gainesville, Florida 32610
Received J uly 9, 1999
The synthesis of four hydroxylated polyamine analogues, (2R,10R)-N1,N11-diethyl-2,10-dihy-
droxynorspermine, (2S,10S)-N1,N11-diethyl-2,10-dihydroxynorspermine, (3S,12S)-N1,N14-diethyl-
3,12-dihydroxyhomospermine, and (3R,12R)-N1,N14-diethyl-3,12-dihydroxyhomospermine, is
described along with their impact on the growth and polyamine metabolism of L1210 murine
leukemia cells. Four different synthetic approaches are set forth, two each for the hydroxylated
norspermines and for the hydroxylated homospermines. The key step in the assembly of the
norspermines was the coupling of either N-[(2R)-2,3-epoxypropyl]-N-ethyl p-toluenesulfonamide
or N-[(2S)-2,3-epoxypropyl]-N-ethyl trifluoromethanesulfonamide to N,N′-dibenzyl-1,3-diami-
nopropane. The key step with homospermines employed alkylation of putrescine with (3S)-N-
(benzyloxycarbonyl)-N-ethyl-3,4-epoxybutylamine or of N,N′-bis(mesitylenesulfonyl)-1,4-
butanediamine with (2R)-2-benzyloxy-4-[N-(mesitylenesulfonyl)ethylamino]-O-tosyl-1-butanol.
All of the hydroxylated analogues were active against L1210 cells with 96-h IC50 values of e2
µM, and they also effectively reduced putrescine and spermidine, although the effect on
spermine pools ranged from moderate to insignificant. Interestingly, the impact of the
hydroxylated analogues on ornithine decarboxylase (ODC) was significantly less than that of
unhydroxylated parent drug (e.g., N1,N11-diethylnorspermine [DENSPM]) at 1 µM; however,
S-adenosylmethionine decarboxylase (AdoMetDC) depletion was nearly identical to what was
observed in cells treated with parent drug. The most notable difference between the parent
and hydroxylated analogues was seen with spermidine/spermine N1-acetyltransferase (SSAT)
upregulation in the DENSPM series. The hydroxylated analogues, especially (R,R)-(HO)2-
DENSPM, were much less effective at upregulation than the parent DENSPM. Finally, a
comparison of the toxicity of (R,R)-(HO)2DENSPM with that of DENSPM at subchronic doses
revealed that the neurological effects seen with DENSPM were now absent.
In tr od u ction
as how they impact on key polyamine biosynthetic
enzymes. For example, while the Ki values of DESPM
and DEHSPM for the polyamine transport system are
within error of each other, the DENSPM value is over
It is now clear that N-alkylated polyamines exhibit
antineoplastic activity against a number of murine and
human tumor lines both in vitro and in vivo.1,2 The
currently ongoing phase II clinical trials with diethyl-
norspermine has further underscored interest in these
molecules. Although it is certainly well-established that
these polyamine analogues utilize the polyamine trans-
port apparatus for incorporation into cells,3,4 deplete
polyamine pools,5 drastically reduce the level of orni-
thine decarboxylase (ODC)6,7 and S-adenosylmethionine
decarboxylase (AdoMetDC) activities,8 and in some
cases upregulate spermidine/spermine N1-acetyltrans-
ferase (SSAT),9-12 the precise mechanism by which they
induce death in tumor cells still remains somewhat of
a mystery.13 A systematic structure-activity study has
shown that very small structural alterations in these
polyamine analogues can cause pronounced changes in
their biological activity at both the cellular and whole
animal level.5 This is exemplified by the differences in
how various analogues compete for transport, as well
10 times as great.14 Although the tetraamines N1,N12
-
diethylspermine (DESPM), N1,N11-diethylnorspermine
(DENSPM), and N1,N14-diethylhomospermine (DEH-
SPM) 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. At a cellular level, the
most profound differences between the three analogues
are related to their ability to stimulate SSAT.10-12 The
tetraamine DENSPM upregulates SSAT by 15-fold in
L1210 murine leukemia cells, while DESPM and DE-
HSPM stimulate SSAT by 4.6- and 1.4-fold, respec-
tively.10 The SSAT stimulation is even more notable in
other cell lines.15,16
At the whole animal level, small structural alterations
also cause a significant difference in pharmacologic
behavior. DEHSPM is a potent antidiarrheal, while
DENSPM has little antitransit activity.17 DEHSPM also
decreases blood pressure in animals, while neither
DENSPM18 nor DESPM possesses this property. In
whole animals, all three analogues are first metabolized
by N-deethylation to the corresponding tetraamine.14,19,20
* To whom correspondence should be addressed. Tel: 352-846-1956.
Fax: 352-392-8406. E-mail: bergeron@mc.cop.ufl.edu.
‡ Parke-Davis Pharmaceutical Research Division, Warner-Lambert
Co., 2800 Plymouth Rd., Ann Arbor, MI 48105.
§ Present address: Albany Molecular Research, Inc., 21 Corporate
Circle, Albany, NY 12203.
10.1021/jm990375z CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/28/1999

Hydroxylated Polyamines as Antiproliferatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 225
Sch em e 1. Synthesis of (R,R)-(HO)2DENSPM (8a )
Using Tosylamide 1a
Sch em e 2. Synthesis of (S,S)-(HO)2DENSPM (8b)
Using Triflamide 9a
a
a
Reagents: (a) (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol,
Reagents: (a) (S)-(-)-4-(chloromethyl)-2,2-dimethyl-1,3-diox-
PPh3, diisopropyl azodicarboxylate, 82%; (b) 1 N HCl, acetone, 95
°C, 87%; (c) TsCl, pyridine, 68%; (d) K2CO3, MeOH, 68%; (e) N,N′-
dibenzyl-1,3-diaminopropane, EtOH, 78 °C, 80%; (f) LiAlH4, THF,
58%; (g) H2, Pd-C, HCl, 68%.
olane, NaH, NaI, 15-crown-5, DMF, 80 °C, 44%; (b) 1 N HCl,
acetone, 95 °C, 93%; (c) TsCl, pyridine, 67%; (d) K2CO3, MeOH,
92%; (e) N,N′-dibenzyl-1,3-diaminopropane, EtOH, 78 °C, 84%; (f)
Na/naphthalene, DME, -78 °C, 50%; (g) H2, Pd-C, HCl, EtOH,
82%.
Sch em e 3. Check of Conservation of Optical Purity for
Both (HO)2DENSPM Synthesesa
Spermine and norspermine are then processed by
deaminopropylation14,19 through the polyamine meta-
bolic network. However, because of the presence of the
aminobutyl fragments in homospermine,20 it is not
terminally N-acylated and cannot be further catabolized.
Thus in animals it remains unmetabolized for a pro-
tracted period of time. These differences in susceptibility
to metabolic degradation correlate to the toxicity pro-
files: DENSPM is the least toxic,19 DEHSPM the
most.20 While DENSPM’s chronic dose-limiting toxicity
is gastrointestinal, its subacute toxicity is neurological.
When treated with 200 mg/kg ip for 6 days, a very high
dose, animals developed severe neurological signs in-
cluding pronounced ataxia, intention tremors, and motor
dysfunction. This dose, with allometric scaling, is nearly
10 times higher than that given to patients. While it
seemed this would be an unlikely problem in the clinic,
it nevertheless represented an easily observable side
effect to help evaluate how structural alterations in the
polyamine backbone impact toxicity. Thus, this paper
focuses on methods for the synthesis of chiral bis-
(hydroxy)polyamine analogues and the effect of this
hydroxylation on the compounds’ activity against L1210
cells in cell culture and on animal toxicity.
Syn th esis
a
Reagents: (a) (R)-(+)-1-phenylethylamine, EtOH, 78 °C, 47%;
Four different hydroxylated tetraamines were syn-
thesized: (R,R)- and (S,S)-N1,N11-diethyl-2,10-dihydroxy-
norspermine [(HO)2DENSPM, 8a in Scheme 1 and 8b
(b) rac-1-phenylethylamine, EtOH, 78 °C, 77%; (c) (R)-(+)-1-
phenylethylamine, EtOH, 78 °C, 79%; (d) rac-1-phenylethylamine,
EtOH, 78 °C, 81%; (e) N-(benzyloxycarbonyloxy)succinimide,
KHCO3, Et2O(aq), 54%; (f) (R)-(-)-Mosher’s acid chloride, pyridine,
100%; (g) e, 72%; (h) f, 94%.
in Scheme 2, respectively] and (S,S)- and (R,R)-N1,N14
-
diethyl-3,12-dihydroxyhomospermine [(HO)2DEHSPM,
29a in Scheme 5 and 29b in Scheme 6, respectively].
The key step in both (HO)2DENSPM cases is the
addition of N,N′-dibenzyl-1,3-diaminopropane21 to 2
equiv of an appropriately substituted epoxide (5 in
Scheme 1 and 13 in Scheme 2). The synthetic sequence
for the preparation of (R,R)-(HO)2DENSPM (8a ) began
with the reaction of tosylamide 1 with (S)-(-)-4-(chlo-
romethyl)-2,2-dimethyl-1,3-dioxolane to produce 2 in

226 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Sch em e 4. Synthesis of Epoxide 23a
Bergeron et al.
Sch em e 6. Synthesis of (R,R)-(HO)2DEHSPM (29b)a
a
Reagents: (a,b) TFAA,34 then BnOH,33 100%; (c,d) N-hydroxy-
succinimide, DCC, then ethylamine in THF, 64%; (e,f) BH3-THF,
then N-(benzyloxycarbonyloxy)succinimide, 33%; (g) TsCl, pyri-
dine, 72%; (h) K2CO3, MeOH, 86%.
Sch em e 5. Synthesis of (S,S)-(HO)2DEHSPM (29a )a
a
Reagents: (a) benzyl 2,2,2-trichloroacetimidate, CF3SO3H,
85%; (b) LiBH4, THF, 83%; (c) TsCl, pyridine, 87%; (d) NaH,
EtNHSO2Mes, DMF, 77%; (e) NaH, MesSO2HN(CH2)4NHSO2Mes,
DMF, 50%; (f) Pd-black, H2, HOAc, H2O, 86%; (g) Na/naphthalene,
DME; (h) EtOH, HCl, 21%.
epoxide 13 (2 equiv) at the less hindered carbon by N,N′-
dibenzyl-1,3-diaminopropane gave the fully protected
tetraamine 14 in 80% yield. An initial attempt to
deprotect 14 with sodium naphthalenide23 gave back
only starting material; therefore, stepwise unmasking
of the nitrogens was employed. Removal of the trifluo-
romethanesulfonyl protective groups in 14 was achieved
by reaction with lithium aluminum hydride,28 giving 7b
in 58% yield. Hydrogenolysis of the internal benzyl
moieties afforded (S,S)-(HO)2DENSPM (8b) as its tet-
rahydrochloride salt (68% yield).
a
Reagents: (a) 23, EtOH; (b) benzyl chloroformate, NEt3,
The conservation of optical purity in both synthetic
sequences was verified in the following manner (Scheme
3). The epoxide 5 was reacted with both racemic and
(R)-1-phenylethylamine, yielding the amino alcohols
CHCl3, 22%; (c) H2, Pd-C, HCl, 85%.
44% yield. Deprotection to the 1,2-diol 3 and selective
monotosylation22 to 4 followed by base-induced ring
closure furnished epoxide 5 in good yield (57% over three
steps). The epoxide 5 was then coupled with N,N′-
dibenzyl-1,3-diaminopropane to give the fully protected
tetraamine 6 (84% yield). Removal of the p-toluene-
sulfonyl groups of 6 was accomplished in 50% yield with
sodium naphthalenide,23 providing 7a . Finally, hydro-
genolytic debenzylation furnished (R,R)-(HO)2DENSPM
(8a ) as its crystalline tetrahydrochloride salt (82%
yield).
1
r a c-15 and 15, respectively. The H NMR spectrum of
15 showed the presence of only one diastereomer upon
comparison with the spectrum of r a c-15. Thus we
concluded that no racemization occurred up to formation
of the epoxide 5. In the same fashion, amino alcohols
r a c-16 and 16 were prepared from the triflamide-
substituted epoxide 13. In this case the 1H NMR spectra
demonstrated that 16 contained about 2% of the dia-
stereomeric compound (as indicated by the doublet at
2.45 ppm with coupling constant of J ) 6.2 Hz). Thus,
epoxide 13 may contain about 2% of the other enanti-
omer.
Chiral integrity was again addressed at the end of
both syntheses (Schemes 1 and 2). The tetraamines 8a
and 8b were derivatized to 17a and 17b, respectively,
by benzyloxycarbonyl groups (CBZs) at their nitrogens
and converted to their respective diastereomeric Mosh-
er’s ester29 derivatives 18a and 18b (Scheme 3). Upon
evaluation of the 1H and 19F NMR spectra, the best
differentiation between 18a and 18b was observed in
the 19F NMR spectra at 45 °C in 12% CDCl3/CD3OD (∆δ
≈ 20 Hz).30 The spectrum of 18a exhibited <2% of the
Alkylation of the ethylamino group with the chiral
three-carbon segment was accomplished by a Mitsunobu
coupling24-26 in the initial step of the synthesis of (S,S)-
(HO)2DENSPM (8b) (Scheme 2). Specifically, reacting
N-ethyltriflamide (9)25 with (S)-(+)-2,2-dimethyl-1,3-
dioxolane-4-methanol gave the dioxolane 10 in 82% yield
(Scheme 2).27 The masking group of the external nitro-
gens of the tetraamine was changed from p-toluene-
sulfonyl to trifluoromethanesulfonyl in order to facilitate
alkylation under Mitsunobu conditions. The steps lead-
ing to 13, analogous to steps b-d in Scheme 1, pro-
ceeded in good yields (40% overall). Ring opening of

Hydroxylated Polyamines as Antiproliferatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 227
Sch em e 7. Check of Conservation of Optical Purity for
signal corresponding to 18b. The NMR of 18b gave a
single peak, in which a similarly minor signal matching
that of 18a could be obscured.31 Thus, both procedures
had successfully produced enantiomeric (HO)2DENSPM
compounds with only minor racemization. A further
indication for the conservation of optical purity through-
out both syntheses is that the absolute values of optical
rotation for the enantiomeric pairs 7a /7b, 8a /8b, and
17a /17b, respectively, are very similar.
Both (HO)2DEHSPM Synthesesa
A synthesis of (R,R)-(HO)2DEHSPM has already been
published from our laboratories.17 Two chiral 4-amino-
2-hydroxybutyl groups were attached to N,N′-diben-
zylputrescine employing (S)-(+)-epichlorohydrin, fol-
lowed by displacement of the chloride by cyanide. The
latter step of this route does not easily lend itself to
scale-up as racemization may occur if the reaction
conditions are not carefully controlled. The mechanism
for this racemization may involve formation of an
azetidinium intermediate in which ring opening by
cyanide anion would lead to epimerization.32
A similar chiral epoxide coupling approach was used
to access N1,N14-diethyl-(3S,12S)-dihydroxyhomosper-
mine [(S,S)-(HO)2DEHSPM, 29a in Scheme 5]. The
synthesis began with the conversion of L-malic acid to
its R-monobenzyl ester 19 (Scheme 4).33-35 Activation
of 19 as its N-hydroxysuccinimide ester and reaction of
this intermediate with ethylamine yielded ethylamide
20 (64%). The reduction of 20 with borane-THF com-
plex followed by protection of the secondary amino group
with CBZ gave 21 in 33% yield. Selective tosylation to
22 and base-promoted ring closure to epoxide 23 com-
pleted the production of this synthon (62% yield over
two steps).
The reaction of epoxide 23 with putrescine (24) and
its N,N′-dibenzylated derivative 25 was investigated
(Scheme 5), and it was found that: (a) both diamines
24 and 25 react with 23 only in protic solvents, i.e.,
alcohols; (b) primary diamine 24 reacts faster than the
more hindered analogue 25; and (c) benzyl derivative
27 reacts to some degree with the alcohol solvent at the
carbobenzyloxy group. For the sake of brevity, we
describe only the optimal conditions: reaction of 24 with
2 equiv of 23 at 55 °C in ethanol, followed by protection
of the amino groups with benzyl chloroformate, gave 28a
in 22% yield over two steps. Hydrogenolysis of all four
protecting groups of 28a produced target tetraamine
29a as its crystalline tetrahydrochloride salt in 85%
yield.
The synthesis of (R,R)-N1,N14-diethyl-3,12-dihydroxy-
homospermine tetrahydrochloride (29b, Scheme 6) was
undertaken to explore an alternative method of access-
ing enantiomerically pure 3,12-(HO)2DEHSPM com-
pounds with the goal of improving the overall yield. This
route (Scheme 6) began with benzylation of the com-
mercially available diester 30 to give the benzyl ether
31,36 followed by reduction using LiBH4 to the diol 32.37
Our yields for these literature preparations were com-
parable (85% and 83%, respectively) to those previously
reported.36,37 The ditosylate intermediate 33 was readily
obtained by derivatization of the diol 32 with p-tolu-
enesulfonyl chloride in pyridine (87% yield).38,39 Alky-
lation at the less hindered site of ditosylate 33 was a
key step in the synthesis of the macrocyclic dihydrox-
amate siderophore alcaligin in these laboratories.39
Selective reaction of 33 at C-4 with the N-ethylmesity-
a
Reagents: (a) (R)-(-)-Mosher’s acid chloride, pyridine, 90%;
(b) N-(benzyloxycarbonyloxy)succinimide, Et2O(aq), 64%; (c) a,
92%.
lenesulfonamide anion to provide synthon 34 in 77%
yield was similarly pivotal to this synthesis. Monoto-
sylate 34 contains a protected ethylamine, the protected
chiral hydroxyl group, and a tosylate leaving group at
C-1. Treatment of 2 equiv of 34 with N,N′-bis(mesity-
lenesulfonyl)-1,4-butanediamine40 (NaH/DMF) resulted
in fully protected intermediate 35 in 50% yield.
Attempts to remove all of the protecting groups of
diether tetrasulfonamide 35 using an alkali metal in
liquid ammonia did not efficiently generate polyamine
29b; thus, a two-step unmasking scheme was employed.
Cleavage of the benzyl ether protecting groups by
catalytic reduction with palladium black41 gave the diol
tetrasulfonamide 36 (86% yield). Finally, conversion of
the four mesitylenesulfonamides to the amines with
sodium naphthalenide23 and acidification gave the
target tetrahydrochloride salt 29b in somewhat low
yield (21%).
The stereochemical integrity of the reaction sequences
to the (HO)2DEHSPMs was verified (Scheme 7). The
precursor 28a was reacted with (R)-(-)-Mosher’s acid
chloride29 to give Mosher’s ester derivative 37a in 90%
yield. In addition, the final polyamine 29b was first
protected as the tetra-CBZ derivative 28b (64% yield)
and subsequently converted to the diastereomeric
Mosher’s ester 37b (92% yield). At 45 °C, the 19F
resonance for 37a was observed at -71.57 ppm and the
signal for 37b appeared, clearly separated, at -71.80
ppm (∆δ ≈ 65 Hz). The spectra of 37a and 37b were
essentially free of resonances indicative of the other
diastereomer.31 Additionally, the specific optical rota-
tions for the enantiomeric compound pairs 28a /28b and
29a /29b gave nearly identical absolute values. Thus,
both routesproducedenantiomericallypure(HO)2DEHSPM
compounds within the limits of detection.
Biologica l Eva lu a tion s
The in vitro biological properties of the hydroxylated
polyamine analogues will be presented in three data
sets: the 48-h and 96-h IC50 values against L1210 cells
and the corresponding Ki values for the polyamine
transport apparatus (Table 1); the effect on polyamine
pools (Table 2); and the impact on ornithine decarboxy-

228 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Bergeron et al.
Ta ble 1. Structures, Abbreviations, L1210 Growth Inhibition, and Transport
a
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
b
variation from the mean typically 10-25% for the 96-h IC50 values. Ki determinations were made by following analogue inhibition of
spermidine transport. All polyamine analogues exhibited simple substrate-competitive inhibition of [3H]SPD transport by L1210 cells.
Values reported represent the mean of at least two or three experiments with a variation typically less than 10%.
Ta ble 2. Impact of Polyamine Analogues on Polyamine Pools
compound
concn (µM) PUTa SPDa SPMa analogueb
DENSPM
Ta ble 3. Effect of Polyamine Analogues on Ornithine
Decarboxylase (ODC), S-Adenosylmethionine Decarboxylase
(AdoMetDC), and Spermidine/Spermine N1-Acetyltransferase
(SSAT) in L1210 Cellsa
10
100
3
30
0
14
6
31
30
1.59
2.44
1.88
1.99
1.18
2.02
0.21
1.89
3.65
2.41
2.43
2.82
3.40
compound
DENSPM
(R,R)-(HO)2DENSPM
(S,S)-(HO)2DENSPM
DEHSPM
ODC
AdoMetDC
SSAT
(R,R)-(HO)2DENSPM
(S,S)-(HO)2DENSPM
DEHSPM
9
5
14
9
46
7
91
40
1
63
54
94
56
110
101
57
7 (10)b
35 (8)
61 (6)
7 (3)
55 (46)
17 (16)
42 (31)
59 (68)
51 (58)
41 (49)
68 (72)
52 (59)
1471
177
816
110
102
114
15
1
5
0.15
0.75
10
66
5
97
49
3
(R,R)-(HO)2DEHSPM
(S,S)-(HO)2DEHSPM
a
(R,R)-(HO)2DEHSPM
(S,S)-(HO)2DEHSPM
40
200
2
5
4
3
3
19
13
11
4
107
104
62
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 reduced ODC to 6.7 ( 2.6% of untreated control;
1 µM DEHSPM reduced AdoMetDC to 40.7 ( 6.2% of untreated
control; and 2 µM DENSPM increased SSAT levels to 1471 ( 120%
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.
10
53
a
Putrescine (PUT), spermidine (SPD), and spermine (SPM)
levels after 48 h of treatment are given as percent polyamine found
in untreated controls. Typical control values in pmol/106 L1210
cells are PUT ) 260 ( 59, SPD ) 3354 ( 361, and SPM ) 658 (
b
119. Analogue amount is expressed as nmol/106 cells. Untreated
L1210 cells (106) correspond to about 1 µL volume; therefore, the
concentration can be estimated as mM.
b
Experiments assessing the impact of increasing the analogue
concentration to 10 µM on ODC and AdoMetDC activity were also
performed on cells treated for 4 and 6 h, respectively. These results
are indicated in parentheses in the table. Data shown represent
the mean of at least three experiments and have variances
consistent with those suggested by the positive control data
presented above.
lase, S-adenosylmethionine decarboxylase, and spermi-
dine/spermine N1-acetyltransferase activities (Table 3).
All tables include not only data on (R,R)- and (S,S)-
(HO)2DENSPM and (R,R)- and (S,S)-(HO)2DEHSPM
but also data on DENSPM and DEHSPM for compari-
son. Finally, a sample of (R,R)-(HO)2DENSPM was
evaluated for neurological toxicity in mice.
IC50 Stu d ies. Unlike DENSPM, both (R,R)- and
(S,S)-(HO)2DENSPM had similar low 48-h and 96-h IC50
values and were thus much more active than the parent
drug at 48 h (Table 1).
The scenario for DEHSPM and its hydroxylated
analogues (R,R)- and (S,S)-(HO)2DEHSPM was quite
the opposite. At 48 h the parent DEHSPM was much
more active than either of the hydroxylated analogues.
Furthermore there was a large difference between the
(R,R)- and (S,S)-(HO)2DEHSPM 48-h IC50 values, al-
though this difference disappeared at 96 h. This differ-
ence between the 48-h and 96-h IC50 is particularly large
(500-fold) with the (R,R)-isomer.
Ki Stu d ies. The ability of the hydroxylated analogues
to compete with radiolabeled SPD for uptake was
evaluated. Both (R,R)- and (S,S)-(HO)2DENSPM had
substantially higher Ki values, 68 and 43 µM, respec-
tively, in comparison to that of DENSPM, 17 µM (Table
1). This was not the case with the hydroxylated homo-
spermines (R,R)- and (S,S)-(HO)2DEHSPM, which had
Ki values of 1.7 and 2.4 µM, essentially the same as that
of the parent drug DEHSPM (1.4 µM) and thus similar
to the spermine Ki of 1.2 µM.
P olya m in e P ools. The following guidelines were
adopted for studying the impact of the analogues on
polyamine pools (Table 2). The measurements were
made after a 48-h exposure to the analogue, and at least
two different concentrations of analogue were evaluated.

Hydroxylated Polyamines as Antiproliferatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 229
Generally, the effect on polyamine pools was evaluated
at the 48-h IC50 concentration and at 5 times this
number.
of AdoMetDC was equally unimpressive, to 41%, with
the same or slightly less reduction in activity seen with
the (R,R)- and (S,S)-(HO)2DEHSPM, 68% and 52%,
respectively. Even raising the analogue concentration
to 10 µM produced little additional effect on AdoMetDC
activity. Finally, while DENSPM had a profound effect
on SSAT, increasing it by 1471%, (S,S)-(HO)2DENSPM
elevated it by 816% and (R,R)-(HO)2DENSPM by only
177%. None of the DEHSPM analogues had any detect-
able impact on SSAT.
At the lower concentrations, the (S,S)-(HO)2DENSPM
differed significantly from the (R,R)-isomer and the
parent DENSPM. (S,S)-(HO)2DENSPM at 1 µM had a
comparatively modest effect on polyamine pools, reduc-
ing putrescine to 66%, spermidine to 46%, and spermine
to 94% of control. At 3 µM the (R,R)-enantiomer
depleted putrescine, spermidine, and spermine to 9%,
14%, and 63% of control values, respectively. At 100 µM
DENSPM, putrescine was depleted to below detectable
limits and spermidine was reduced to around 6% of
controls, while spermine levels were diminished to 30%.
At 15 µM (R,R)-(HO)2DENSPM, putrescine was reduced
to 5% of control, spermidine to 9%, and spermine to 54%.
At 5 µM (S,S)-analogue, native polyamines were reduced
to the same extent within experimental error. Finally,
at 5 times the IC50 both (R,R)- and (S,S)-(HO)2DENSPM
achieved similar intracellular levels of 2 mM, as did
DENSPM itself.
In Vivo Toxicit y. Clinically, DENSPM has so far
presented with minimal side effects. However, we have
shown that at very high subchronic doses, neurological
side effects can be induced in mice. When 12 male C-57/
Bl/6 mice were treated with DENSPM tetrahydrochlo-
ride at a dose of 200 mg/kg/day for 6 days, all of the
animals displayed severe neurological signs during the
course of treatment. These signs included severe ataxia,
intention tremors, and motor dysfunction. By day 3, 2
of the animals died of seizures and by day 6 a total of
10 of the 12 animals had died from neurological se-
quelae. In a parallel experiment, for 6 days 12 mice were
given an equimolar dose (217 mg/kg/day ip) of (R,R)-
(HO)2DENSPM tetrahydrochloride with a lower ee
value than the current compound. None of these 12
animals presented with any neurological symptoms at
all. None of the animals died, nor was there weight loss
or any other adverse effect. When this experiment was
repeated with three mice given enantiomerically pure
(R,R)-(HO)2DENSPM, again virtually no signs of toxic-
ity were observed. Thus it seems that hydroxylation of
diethylnorspermine, while actually increasing the par-
ent compound’s antiproliferative activity in cell culture
at 48 h, at the same time markedly decreases the in
vivo toxicity. Acute and chronic studies of both (R,R)-
and (S,S)-(HO)2DEHSPM as well as both (R,R)- and
(S,S)-(HO)2DENSPM are now underway.
Although DEHSPM has a much lower 48-h IC50 value
than DENSPM, its impact on the polyamine pools even
at 0.75 µM, 5 times the 48-h IC50, is much less
significant. Putrescine was reduced to 49% and sper-
midine to 40%, and spermine was unchanged. However,
higher concentrations of DEHSPM (e.g., 10 µM) were
shown to reduce the polyamine levels at 48 h to well
below this. At 5 times the 48-h IC50 value, 200 µM (R,R)-
(HO)2DEHSPM reduced putrescine to 4% of control and
spermidine to 13% with essentially no impact on sper-
mine. At 10 µM (S,S)-(HO)2DEHSPM, again 5 times the
48-h IC50 concentration, putrescine and spermidine were
reduced to 3% and 4% of control and spermine to 53%
of control. At the 48-h IC50 concentrations, 40 µM (R,R)-
(HO)2DEHSPM and 2 µM (S,S)-(HO)2DEHSPM both
reduced putrescine to below 5% and spermidine to below
19%. The (R,R)-isomer had no effect on spermine while
the (S,S)-isomer reduced it to 62% of control. Finally,
at 5 times the IC50 value the (S,S)-enantiomer achieved
slightly higher concentrations intracellularly than the
(R,R)-isomer, 3.4 mM versus 2.4 mM.
Discu ssion
Synthetic routes to (R,R)-(HO)2DENSPM, (S,S)-
(HO)2DENSPM, (R,R)-(HO)2DEHSPM, and (S,S)-(HO)2-
DEHSPM have been successfully executed, and the
chiral integrity of the schemes was verified. Four
separate synthetic approaches were evaluated, one for
each of the hydroxylated analogues. Both routes to
(HO)2DENSPM consisted of seven steps and utilized the
same central fragment, N,N′-dibenzyl-1,3-diaminopro-
pane, but different chiral epoxides, one a triflamide and
the other a tosylamide. The Mitsunobu coupling of
N-ethyltriflamide (9) with the chiral dioxolane carbinol,
leading to the chiral synthon 10, was more efficient than
alkylation of N-ethyltosylamide (1) with the chloro-
methyldioxolane. We did observe a minor amount of the
(R)-enantiomer (e2%) in the production of (S)-triflamide
epoxide 13 on the route to (S,S)-(HO)2DENSPM (8b).
We do not regard this level of impurity as a significant
problem. No such racemization was detected in making
tosylamide epoxide 5 in the route to (R,R)-(HO)2-
DENSPM. The Mosher’s ester derivatives (18a and 18b)
of the (HO)2DENSPMs confirmed that the level of
racemization was not problematic.
Im p a ct of An a logu es on ODC, Ad oMetDC, a n d
SSAT. As previously described, there is little correlation
between the ODC and AdoMetDC levels and a polyamine
analogue’s 48-h and 96-h IC50 values.40 At 4 h, 1 µM
DENSPM reduced ODC activity to 7% of control, while
(R,R)- and (S,S)-(HO)2DENSPM lowered ODC to 35%
and 61% of control, respectively (Table 3). However,
increasing the analogue concentration to 10 µM resulted
in a marked decrease in ODC activity, to 8% and 6% of
control values, by (R,R)- and (S,S)-(HO)2DENSPM,
respectively. Diethylhomospermine was as active as
DENSPM at 1 µM, lowering ODC to 7% of control.
Again, both hydroxylated compounds were less active
at 1 µM with a reduction to 55% and 17% of control ODC
by (R,R)- and (S,S)-(HO)2DEHSPM, respectively. In
contrast to the finding with the hydroxylated DENSPM
analogues, raising the concentration of the hydroxylated
DEHSPM analogues to 10 µM did not result in a
dramatic further reduction of ODC activity. Diethyl-
norspermine, (R,R)-(HO)2DENSPM, and (S,S)-(HO)2-
DENSPM reduced AdoMetDC to 42%, 59%, and 51%,
respectively, of control. Diethylhomospermine reduction
The two synthetic routes leading to the longer hy-
droxylated tetraamines (S,S)- and (R,R)-(HO)2DEHSPM
(29a and 29b, respectively) began with the appropriate

230 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Bergeron et al.
(-)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane (11.00 g, 9.4
mL, 75.3 mmol) was added dropwise. The reaction mixture
was kept at this temperature for 70 h, then cooled to room
temperature, and poured onto ice-cold water (500 mL). The
aqueous layer was extracted with EtOAc (3 × 150 mL). The
combined organic layers were washed with saturated NaCl
solution (100 mL), dried over MgSO4, and concentrated in
vacuo. The resulting oil was purified by flash chromatography
(15% EtOAc/cyclohexane) to give 2 (6.87 g, 44%) as a colorless
oil: 1H NMR (CDCl3) δ 1.11 (t, 3 H, J ) 7.3), 1.33 (s, 3 H),
1.40 (s, 3 H), 2.42 (s, 3 H), 3.10 (dd, 1 H, J ) 14.5, 6.2), 3.17-
3.47 (m, 3 H), 3.78 (dd, 1 H, J ) 8.6, 6.4), 4.09 (dd, 1 H, J )
8.6, 6.2), 4.29 (m, 1 H), 7.30 (d, 2 H, J ) 8.1), 7.70 (m, 2 H);
HRMS m/z calcd for C15H24NO4S 314.1426, found 314.1425;
malic acid. Its L-enantiomer was transformed into
N-CBZ-N-ethyl-N-epoxybutyl synthon 23, which was
reacted with putrescine to assemble the polyamine
framework of 29a . Our alternate route to the enantio-
meric (HO)2DEHSPM (29b) began with commercially
available dimethyl D-malate (30), which was converted
into the chiral tosylate 34. Bis-alkylation of N,N′-
bis(mesitylenesulfonyl)-1,4-butanediamine with 34
completed the assembly of the skeleton of (R,R)-
(HO)2DEHSPM (29b). Chiral integrity was maintained
throughout both routes to the dihydroxylated homo-
spermines, as confirmed by 19F NMR spectral analysis
of the appropriate Mosher’s ester derivatives 37a and
37b.
At a cellular level in vitro, the most notable difference
between the polyamine analogues DENSPM and DE-
HSPM and their hydroxylated counterparts occurred at
the onset of growth inhibition and impact on polyamine
enzymes. The hydroxylated DENSPM analogues inhib-
ited growth faster than DENSPM, while the hydroxy-
lated DEHSPM analogues were slower in onset than
DEHSPM. Under our standard experimental conditions,
reduction in ODC activity by all of the hydroxylated
analogues was less than that seen with the parent
drugs. However, suppression of AdoMetDC was very
much like that observed with the parents. The most
notable difference in alteration of polyamine enzyme
activity was associated with SSAT and the hydroxylated
DENSPMs. The upregulation of SSAT induced by (S,S)-
(HO)2DENSPM was 55% of that seen with DENSPM,
while (R,R)-(HO)2DENSPM upregulated SSAT only
12% as effectively as DENSPM. Finally, at the whole
animal level in vivo there was a remarkable differ-
ence in neurotoxicity between DENSPM and (R,R)-
(HO)2DENSPM. At equimolar doses, the parent
DENSPM is uniformly neurotoxic, while the hydroxy-
lated analogue is virtually nontoxic. This suggests that
hydroxylation may be a viable approach to altering the
toxicity profile of polyamine analogues. However, fur-
ther long-term chronic toxicity trials are necessary.
[R]22 +14.1 (c 0.95, CHCl3). Anal. (C15H23NO4S) C, H, N.
D
N-[(2R)-2,3-Dih ydr oxypr opyl]-N-eth yl-p-tolu en esu lfon a-
m id e (3). Hydrochloric acid (1 N, 40 mL) was added to a
solution of 2 (6.60 g, 21.1 mmol) in acetone (20 mL). The
reaction mixture was heated to 95 °C for 1 h, then cooled to
room temperature, and concentrated in vacuo. Purification by
flash chromatography (30% cyclohexane/EtOAc) gave 3 (5.33
g, 93%) as an oil: 1H NMR (CDCl3) δ 1.09 (t, 3 H, J ) 7.1),
2.43 (s, 3 H), 3.06 (dd, 1 H, J ) 14.5, 7.3), 3.16-3.40 (m, 3 H),
3.53 (m, 2 H), 3.81 (m, 1 H), 7.38 (m, 2 H), 7.72 (m, 2 H); HRMS
m/z calcd for C12H20NO4S 274.1113, found 274.1114; [R]22D -1.0
(c 0.99, CHCl3).
N-Eth yl-N-[(2R)-2-h ydr oxy-3-p-tolu en esu lfon atopr opyl]-
p-tolu en esu lfon a m id e (4). A solution of 3 (5.74 g, 21.0 mmol)
in anhydrous pyridine (50 mL) was cooled to 0 °C under an
Ar atmosphere, and TsCl (4.41 g, 23.1 mmol) was added in
portions. The reaction mixture was maintained at 4 °C for 22
h, diluted with Et2O (500 mL), and extracted with 1 N HCl (4
× 50 mL). The organic phase was dried over MgSO4, concen-
trated in vacuo, and purified by flash chromatography (5%
acetone/CHCl3) to yield 4 (6.00 g, 67%) as a white solid: mp
1
110-112 °C; H NMR (CD3OD) δ 1.01 (t, 3 H, J ) 7.2), 2.42
(s, 3 H), 2.46 (s, 3 H), 2.99 (m, 1 H), 3.05-3.30 (m, 3 H), 3.94
(m, 2 H), 4.09 (m, 1 H), 7.37 (m, 2 H), 7.45 (d, 2 H, J ) 8.6),
7.6 (d, 2 H, J ) 8.4), 7.81 (d, 2 H, J ) 8.4); [R]23 -10.1 (c
D
1.01, CHCl3). Anal. (C19H25NO6S2) C, H, N.
N-[(2R)-2,3-E p oxyp r op yl]-N-et h yl-p -t olu en esu lfon a -
m id e (5). To a solution of 4 (5.89 g, 13.8 mmol) in CH3OH
(100 mL) was added K2CO3 (2.02 g, 14.6 mmol). The suspen-
sion was stirred at room temperature for 3 h under an Ar
atmosphere and then was poured into a mixture of H2O (150
mL) and CH2Cl2 (150 mL). Saturated NaCl solution was added,
and the layers were separated. The aqueous layer was
extracted with CH2Cl2 (3 × 100 mL). The combined organic
phase was dried over MgSO4 and concentrated in vacuo.
Purification by flash chromatography (20% EtOAc/cyclohex-
ane) gave 5 (3.24 g, 92%) as a colorless oil: 1H NMR (CDCl3)
δ 1.16 (t, 3 H, J ) 7.0), 2.42 (s, 3 H), 2.55 (dd, 1 H, J ) 4.6,
2.4), 2.79 (t, 1 H, J ) 4.6), 2.97 (dd, 1 H, J ) 14.9, 6.4), 3.08
(m, 1 H), 3.26 (m, 1 H), 3.34 (m, 1 H), 3.63 (dd, 1 H, J ) 15.2,
3.5), 7.30 (d, 2 H, J ) 7.9), 7.70 (m, 2 H); 13C NMR (CDCl3 )
77.0 ppm) δ 13.91, 21.45, 43.89, 45.17, 50.04, 50.90, 127.03,
129.71, 136.84, 143.33; HRMS m/z calcd for C12H18NO3S
Exp er im en ta l Section
Gen er a l. Reagents were purchased from the Aldrich, Fluka,
or Sigma Chemical Co. and were used without further puri-
fication. Fisher Optima-grade solvents were routinely used.
DMF and THF were distilled, the latter from sodium and
benzophenone. Silica gel 32-60 (40 µm “flash”) from Selecto,
Inc. (Kennesaw, GA) was used for flash column chromatogra-
phy. NMR spectra were recorded on a Varian Unity 300 with
1H NMR spectra at 300 MHz, 13C NMR spectra at 75 MHz,
and 19F NMR spectra at 282 MHz. Chemical shifts are given
in parts per million downfield from an internal tetramethyl-
silane standard, unless otherwise specified. Coupling constants
(J ) are in Hz. Optical rotations were measured at 589 nm
(sodium D line) unless otherwise indicated with a Perkin-Elmer
341 polarimeter; c equals grams of compound per 100 mL of
solution. Melting points were determined on a Fisher-J ohns
melting point apparatus and are uncorrected. FAB mass
spectra were run in a 3-nitrobenzyl alcohol or glycerol matrix.
Elemental analyses were performed by Atlantic Microlabs,
Norcross, GA.
256.1007, found 256.1047; [R]24 +21.4 (c 0.95, CHCl3). Anal.
D
(C12H17NO3S) C, H, N.
(2S,10S)-N1,N11-Bis(p-tolu en esu lfon yl)-N4,N8-d iben zyl-
N1,N11-d ieth yl-2,10-d ih yd r oxyn or sp er m in e (6). A solution
of 5 (3.09 g, 12.1 mmol) and N,N′-dibenzyl-1,3-diaminopropane
(1.54 g, 6.06 mmol) in EtOH (50 mL) was heated to reflux for
19 h under an Ar atmosphere. The solvent was removed under
reduced pressure, and the residue was purified by flash
chromatography (5% then 10% acetone/CHCl3) to give 6 (3.89
g, 84%) as a colorless, viscous oil: 1H NMR (CD3OD) δ 1.02 (t,
6 H, J ) 7.0), 1.70 (m, 2 H), 2.40 (s, 6 H), 2.42 (m, 4 H), 2.48
(m, 4 H), 2.76 (dd, 2 H, J ) 14.7, 8.1), 3.20 (m, 2 H), 3.26 (m,
2 H), 3.36 (dd, 2 H, J ) 14.7, 3.5), 3.46 (d, 2 H, J ) 13.4), 3.63
(d, 2 H, J ) 13.4), 3.84 (m, 2 H), 7.12-7.30 (m, 10 H), 7.34 (d,
4 H, J ) 7.9), 7.67 (m, 4 H); 13C NMR (CDCl3 ) 77.0 ppm) δ
13.89, 21.46, 24.36, 44.61, 51.75, 52.12, 57.69, 58.88, 66.97,
127.13, 127.25, 128.41, 129.06, 129.63, 136.77, 138.33, 143.15;
N-[(4R)-2,2-Dim eth yl-1,3-d ioxola n -4-ylm eth yl]-N-eth yl-
p-tolu en esu lfon a m id e (2). A solution of 1 (10.00 g, 50.2
mmol) in DMF (90 mL) was cooled to 0 °C under an Ar
atmosphere, and 60% NaH (3.00 g, 75.3 mmol) was carefully
added. The reaction mixture was warmed to room tempera-
ture, and NaI (375 mg, 2.5 mmol) and 15-crown-5 (550 mg,
495 µL, 2.5 mmol) were added. After heating to 80 °C, (S)-

Hydroxylated Polyamines as Antiproliferatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 231
HRMS m/z calcd for C41H57N4O6S2 765.3720, found 765.3721;
N-E t h yl-N-[(2S)-2-h yd r oxy-3-p -t olu en esu lfon a t op r o-
p yl]tr iflu or om eth a n esu lfon a m id e (12). p-Tosyl chloride
(1.90 g, 9.98 mmol) was introduced in portions to 11 (2.28 g,
9.08 mmol) in pyridine (20 mL) at 0 °C under an Ar atmo-
sphere. The reaction mixture was stirred for 5.5 h, diluted with
Et2O (200 mL), and extracted with 1 N HCl (4 × 50 mL). The
organic layer was dried over MgSO4, concentrated in vacuo,
and purified by flash chromatography (10%, 20%, then 50%
EtOAc/cyclohexane) to furnish 12 (2.50 g, 68%) as a colorless
oil: 1H NMR (CDCl3) δ 1.26 (t, 3 H, J ) 7.1), 2.47 (s, 3 H),
2.55 (d, 1 H, J ) 4.8), 3.28-3.64 (m, 4 H), 3.96-4.17 (m, 3 H),
7.38 (m, 2 H), 7.80 (m, 2 H); HRMS m/z calcd for C13H19F3-
[R]24 -27.8 (c 1.06, CHCl3).
D
(2R,10R)-N4,N8-Diben zyl-N1,N11-dieth yl-2,10-dih ydr oxy-
n or sp er m in e (7a ). Anhydrous 1,2-dimethoxyethane (DME;
75 mL) was added dropwise to a mixture of Na (2.46 g, 107
mmol) and naphthalene (16.88 g, 132 mmol)23 under an Ar
atmosphere. The reaction mixture was stirred for 2 h at room
temperature to give a dark green solution. A portion of this
solution was added dropwise to 6 (3.56 g, 4.65 mmol) in DME
(150 mL) at -78 °C until a green color persisted. The reaction
mixture was quenched with saturated NaHCO3 (8 mL) and
warmed to room temperature. Solid K2CO3 (50 g) was added,
and the mixture was stirred for 2 h. The solids were filtered
off and washed with Et2O (3 × 100 mL). The filtrate was
concentrated in vacuo and purified by flash chromatography
(CH3OH; 5% then 10% concentrated NH4OH/CH3CN) to give
7a (1.06 g, 50%) as a viscous oil: 1H NMR (CD3OD) δ 1.09 (t,
6 H, J ) 7.3), 1.70 (m, 2 H), 2.33-2.70 (m, 16 H), 3.51 (d, 2 H,
J ) 13.4), 3.63 (d, 2 H, J ) 13.4), 3.79 (m, 2 H), 7.10-7.45 (m,
10 H); HRMS m/z calcd for C27H45N4O2 457.3543, found
NO6S2 406.0606, found 406.0603; [R]25 -5.0 (c 1.04, CHCl3).
D
Anal. (C13H18F3NO6S2) C, H, N.
N-[(2S)-2,3-E p oxyp r op yl]-N-et h ylt r iflu or om et h a n e-
su lfon a m id e (13). According to the method described for
compound 5, 12 was reacted with K2CO3 (915 mg, 6.62 mmol)
in CH3OH (20 mL). Purification by flash chromatography (10%
EtOAc/cyclohexane) generated 13 (949 mg, 68%) as a colorless
oil: 1H NMR (CDCl3) δ 1.30 (t, 3 H, J ) 7.1), 2.58 (dd, 1 H, J
) 4.5, 2.2), 2.86 (t, 1 H, J ) 4.5), 3.08 (br m, 1 H), 3.15 (m, 1
457.3549; [R]25 -65.8 (c 1.12, CHCl3).
D
H), 3.59 (m, 2 H), 3.92 (m, 1 H); [R]24 -35.0 (c 0.98, CHCl3).
(2R,10R)-N1,N11-Diet h yl-2,10-d ih yd r oxyn or sp er m in e
Tetr a h yd r och lor id e (8a ). Hydrochloric acid (1 N, 17.5 mL,
17.5 mmol) and 10% Pd-C (200 mg) were added to 7a (1.00 g,
2.19 mmol) in EtOH (50 mL). The reaction mixture was stirred
under H2 at 1 atm for 22 h, then filtered through Celite (0.5
cm), and washed with water (50 mL) and EtOH (50 mL). The
solvents were removed in vacuo. The residue was taken up in
water (15 mL), filtered through a syringe filter (0.2 µm, PVDF),
and again concentrated. Recrystallization from aqueous EtOH
gave 8a (761 mg, 82%) as small white crystals: 1H NMR (D2O,
NaTSP) δ 1.32 (t, 6 H, J ) 7.2), 2.14 (m, 2 H), 3.03-3.30 (m,
16 H), 4.27 (tt, 2 H, J ) 9.6, 3.2); 13C NMR (D2O; dioxane )
67.19 ppm) δ 10.99, 23.00, 43.92, 45.36, 49.99, 50.72, 63.52;
HRMS m/z calcd for C13H33N4O2 277.2604, found 277.2578;
D
Anal. (C6H10F3NO2S) C, H, N.
(2R,10R)-Bis(t r iflu or om et h a n esu lfon yl)-N4,N8-d ib en -
zyl-N1,N11-dieth yl-2,10-dih ydr oxy-N1,N11-n or sper m in e (14).
Compound 13 (823 mg, 3.53 mmol) was reacted with N,N′-
dibenzyl-1,3-diaminopropane (449 mg, 1.76 mmol) by the
method used to synthesize 6. Purification by flash chroma-
tography (10% acetone/CHCl3) provided 14 (1.02 g, 80%) as a
colorless, viscous oil: 1H NMR (CD3OD) δ 1.19 (t, 6 H, J )
7.1), 1.69 (quint, 2 H, J ) 7.2), 2.40 (d, 4 H, J ) 6.6), 2.49 (m,
4 H), 3.00 (br m, 2 H), 3.45 (d, 2 H, J ) 13.1), 3.65 (d, 2 H, J
) 13.1), 3.40-3.73 (m, 6 H), 3.85 (m, 2 H), 7.12-7.43 (m, 10
H); HRMS m/z calcd for C29H43F6N4O6S2 721.2528, found
721.2519; [R]24D +31.0 (c 1.05, CHCl3). Anal. (C29H42F6N4O6S2)
C, H, N.
[R]24 -2.1 (c 0.75, 1 N HCl). Anal. (C13H36Cl4N4O2) C, H, Cl,
D
(2S,10S)-N4,N8-Diben zyl-N1,N11-d ieth yl-2,10-d ih yd r oxy-
n or sp er m in e (7b). A solution of LiAlH4 (1.0 M in THF, 3.72
mL, 3.72 mmol) was carefully added to 14 (448 mg, 0.62 mmol)
in THF (30 mL) at room temperature under an Ar atmosphere.
The reaction mixture was heated to reflux for 19 h, cooled to
room temperature, and carefully hydrolyzed with H2O (435
µL), 15% NaOH (435 µL), and H2O (1.3 mL). The solids were
filtered and washed with Et2O (2 × 5 mL). The filtrate was
concentrated in vacuo and was purified by flash chromatog-
raphy (5% then 10% concentrated NH4OH/CH3CN). The
product was dissolved in CH3OH (5 mL), filtered through a
syringe filter (0.2 mm, PVDF), and concentrated in vacuo to
give 7b (165 mg, 58%) as a viscous oil: 1H NMR (CD3OD) δ
1.10 (t, 6 H, J ) 7.1), 1.70 (m, 2 H), 2.34-2.70 (m, 16 H), 3.52
(d, 2 H, J ) 13.4), 3.62 (d, 2 H, J ) 13.4), 3.79 (m, 2 H), 7.10-
7.45 (m, 10 H); HRMS m/z calcd for C27H45N4O2 457.3543,
N.
N-E t h ylt r iflu or om et h a n esu lfon a m id e (9). Trifluoro-
methanesulfonic anhydride (10 g, 35 mmol) in CH2Cl2 (20 mL)
was added dropwise over 20 min to ethylamine in THF (2.0
M, 51 mL, 102 mmol) in CH2Cl2 (100 mL) at -78 °C under an
Ar atmosphere. The reaction mixture was stirred for 2.5 h,
warmed to room temperature, and washed with 1 N HCl (2 ×
100 mL). The organic phase was dried over MgSO4 and
concentrated in vacuo. Distillation (bp 60 °C/ca. 2 mmHg) [lit.
bp 95-97 °C/0.3 mmHg]25 gave 9 (3.35 g, 54%) as a colorless
oil: 1H NMR (CDCl3) δ 1.29 (t, 3 H, J ) 7.3), 3.38 (m, 2 H),
4.76 (m br, 1 H); HRMS (EI) m/z calcd for C3H6F3NO2S
177.0071, found 177.0028.
N-[(4S)-2,2-Dim eth yl-1,3-d ioxola n -4-ylm eth yl]-N-eth yl-
tr iflu or om eth a n esu lfon a m id e (10). (S)-(+)-2,2-Dimethyl-
1,3-dioxolane-4-methanol (1.47 mL, 1.57 g, 11.9 mmol) and
triphenylphosphine (3.75 g, 14.3 mmol) were added to 9 (2.11
g, 11.9 mmol) in THF (50 mL) under an Ar atmosphere.
Diisopropyl azodicarboxylate (2.81 mL, 2.89 g, 14.3 mmol) was
added dropwise at room temperature, and the reaction mixture
was stirred for 4.5 h. The solvent was removed in vacuo, and
the crude product was purified by flash chromatography (15%
EtOAc/cyclohexane) to give 10 (2.83 g, 82%) as a colorless oil:
1H NMR (CDCl3) δ 1.28 (t, 3 H, J ) 7.3), 1.35 (s, 3 H), 1.42 (s,
3 H), 3.33 (m, 1 H), 3.57-3.65 (m, 3 H), 3.66 (dd, 1 H, J ) 8.6,
6.4), 4.11 (dd, 1 H, J ) 8.6, 6.4), 4.31 (m, 1 H); HRMS m/z
calcd for C9H17F3NO4S 292.0830, found 292.0853; [R]24D -23.1
(c 1.02, CHCl3). Anal. (C9H16F3NO4S) C, H, N.
found 457.3547; [R]26 +62.1 (c 1.10, CHCl3).
D
(2S,10S)-N1,N11-Dieth yl-2,10-dih ydr oxyn or sper m in e Tet-
r a h yd r och lor id e (8b). Compound 7b (162 mg, 0.35 mmol)
in 1 N HCl (2.84 mL) and EtOH (10 mL) was stirred under H2
(1 atm) as described for the synthesis of 8a . Recrystallization
from aqueous EtOH gave 8b (101 mg, 68%) as small white
crystals: 1H NMR (D2O, NaTSP) δ 1.32 (t, 6 H, J ) 7.3), 2.13
(m, 2 H), 3.03-3.28 (m, 16 H), 4.27 (tt, 2 H, J ) 9.6, 3.2); 13C
NMR (D2O; dioxane ) 67.19 ppm) δ 11.02, 23.00, 43.93, 45.37,
49.99, 50.71, 63.50; HRMS m/z calcd for C13H33N4O2 277.2604,
found 277.2601; [R]24 +1.8 (c 0.77, 1 N HCl). Anal. (C13H36
-
D
Cl4N4O2) C, H, Cl, N.
N-[(2S)-2,3-Dih ydr oxypr opyl]-N-eth yltr iflu or om eth an e-
su lfon a m id e (11). A solution of 10 (2.81 g, 9.65 mmol) in
acetone (20 mL) and 1 N HCl (40 mL) was heated to reflux for
1 h. The solvent was removed in vacuo, and the residue was
purified by flash chromatography (60% EtOAc/cyclohexane) to
give 11 (2.12 g, 87%) as an oil: 1H NMR (CD3OD) δ 1.27 (t, 3
H, J ) 7.0), 3.20-3.40 (m, 1 H), 3.46-3.74 (m, 5 H), 3.85 (m,
1 H); HRMS m/z calcd for C6H13F3NO4S 252.0517, found
N-E t h yl-N-[(2S)-2-h yd r oxy-3-[(1R)-1-p h en ylet h yla m i-
n o]p r op yl]-p-tolu en esu lfon a m id e (15). A solution of 5 (23
mg, 91 µmol) and (R)-(+)-1-phenylethylamine (22 mg, 23
µL, 183 µmol) in EtOH (5 mL) was heated to reflux for
17 h under an Ar atmosphere. Solvent was removed, and
the crude product was purified by flash chromatography
(50% EtOAc/cyclohexane, then EtOAc) to give 15 (16 mg,
47%) as a colorless oil: 1H NMR (CD3OD) δ 1.01 (t, 3
H, J ) 7.2), 1.37 (d, 3 H, J ) 6.7), 2.42 (s, 3 H), 2.43
(dd, 1 H, J ) 12.3, 7.7), 2.54 (dd, 1 H, J ) 12.3, 4.4),
252.0558; [R]24 -6.0 (c 1.00, CHCl3). Anal. (C6H12F3NO4S) C,
D
H, N.

232 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Bergeron et al.
3.03 (dd, 1 H, J ) 14.5, 6.8), 3.11-3.28 (m, 3 H), 3.76
(q, 1 H, J ) 6.7), 3.83 (m, 1 H), 7.15-7.35 (m, 5 H),
CD3OD, CFCl3, 45 °C) δ -71.20; HRMS m/z calcd for
C
65H71F6N4O14 1245.4871, found 1245.4818; [R]26 -27.2 (c
D
7.36 (m,
2
H), 7.67 (m,
2
H); HRMS m/z calcd for
1.68, CHCl3).
20H29N2O3S 377.1899, found 377.1932; [R]24 +28.7 (c 0.78,
(2S,10S)-N1,N11-Diet h yl-2,10-d ih yd r oxy-N1,N4,N8,N11
-
C
D
CHCl3).
tetr a k is(ben zyloxyca r bon yl)n or sp er m in e (17b). Accord-
ing to the procedure described for the preparation of 17a , 8b
(37 mg, 89 µmol) was reacted with KHCO3 (233 mg, 2.32 mmol)
and N-(benzyloxycarbonyloxy)succinimide (132 mg, 0.53 mmol).
Purification of the crude product by flash chromatography
(10%, then 25% acetone/CHCl3) gave 17b (52 mg, 72%) as a
colorless oil: 1H NMR (CD3OD) δ 0.95-1.20 (m, 6 H), 1.75 (m,
2 H), 2.80-3.60 (m, 16 H), 3.96 (m, 2 H), 4.95-5.20 (m, 8 H),
7.10-7.50 (m, 20 H); HRMS m/z calcd for C45H57N4O10 813.4075,
N-Eth yl-N-[(2S)-2-h yd r oxy-3-(1-p h en yleth yla m in o)p r o-
p yl]-p-tolu en esu lfon a m id e (r a c-15). A solution of 5 (19 mg,
76 µmol) and (()-1-phenylethylamine (18 mg, 152 µmol) in
EtOH (5 mL) was heated to reflux for 17 h under an Ar
atmosphere. The reaction mixture was concentrated in vacuo,
and the crude product was purified by flash chromatography
(33% EtOAc/cyclohexane, then EtOAc) to yield r a c-15 (22 mg,
77%) as a colorless oil: 1H NMR (CD3OD) δ 1.01 (t, 3 H, J )
7.2), 1.03 (t, 3 H, J ) 7.3), 1.37 (d, 6 H, J ) 6.7), 2.33 (dd, 1 H,
J ) 12.3, 8.4), 2.42 (s, 6 H), 2.43 (dd, 1 H, J ) 12.3, 7.7), 2.54
(dd, 1 H, J ) 12.3, 4.4), 2.64 (dd, 1 H, J ) 12.3, 3.7), 2.96-
3.30 (m, 8 H), 3.76 (q, 2 H, J ) 6.7), 3.83 (m, 1 H), 3.90 (m, 1
H), 7.17-7.34 (m, 10 H), 7.38 (m, 4 H), 7.67 (m, 4 H).
found 813.4083; [R]27 +4.7 (c 1.01, 1 N HCl).
D
(2S,10S)-2,10-Bis[(S)-r-m et h oxy-r-(t r iflu or om et h yl)-
p h en yla cetoxy]-N1,N11-d ieth yl-N1,N4,N8,N11-tetr a k is(ben -
zyloxyca r bon yl)n or sp er m in e (18b). According to the pro-
cedure given for the synthesis of 18a , 17b (51 mg, 69 µmol)
was reacted with (R)-(-)-Mosher’s acid chloride (38 µL, 176
µmol) to furnish 18b (65 mg, 94%) as a colorless oil: 1H NMR
(CDCl3, 45 °C) δ 0.96 (m, 6 H), 1.55-1.75 (m, 2 H), 3.41 (s, 3
H), 2.85-3.65 (m, 16 H), 4.90-5.20 (m, 8 H), 5.43 (m, 2 H),
7.22-7.48 (m, 30 H); 19F NMR (CDCl3, CFCl3, 45 °C) δ -71.81;
19F NMR (12% CDCl3/CD3OD, CFCl3, 45 °C) δ -71.27; HRMS
N-Eth yl-N-[(2R)-2-h yd r oxy-3-[(1R)-1-p h en yleth yla m i-
n o]p r op yl]tr iflu or om eth a n esu lfon a m id e (16). By the pro-
cedure used for the preparation of 15, 13 (19 mg, 82.3 µmol)
was reacted with (R)-(+)-1-phenylethylamine (20 mg, 165
µmol) and purified by flash chromatography (5% CH3OH/
CHCl3) to give 16 (23 mg, 79%) as a colorless oil: 1H NMR
(CD3OD) δ 1.22 (t, 3 H, J ) 7.1), 1.37 (d, 3 H, J ) 6.8), 2.34
(dd, 1 H, J ) 12.3, 8.1), 2.56 (dd, 1 H, J ) 12.3, 4.0), 3.28-
3.44 (m, 2 H), 3.57 (m, 1 H), 3.76 (q, 1 H, J ) 6.6), 3.92 (m, 1
H), 7.15-7.35 (m, 5 H); HRMS m/z calcd for C14H22F3N2O3S
m/z calcd for C65H71F6N4O14 1245.4871, found 1245.4852; [R]26
-13.6 (c 1.64, CHCl3). Anal. (C65H70F6N4O14) C, H, N.
D
Ben zyl (2S)-4-(Eth yla m in o)-2-h yd r oxy-4-oxobu ta n oa te
(20). N-Hydroxysuccinimide (6.83 g, 59.4 mmol) and DCC
(12.25 g, 59.4 mmol) were added to 19 (12.10 g, 54.0 mmol) in
CH2Cl2 (300 mL) under an Ar atmosphere at 0 °C. The ice bath
was removed after 30 min, and the reaction mixture was
stirred at room temperature for an additional 4 h. Solids were
filtered and washed with CH2Cl2, and the solvent was removed
in vacuo. The residue was dissolved in THF (100 mL) and
cooled to 0 °C, and ethylamine in THF (2.0 M, 27 mL, 54 mmol)
was added dropwise. The reaction mixture was stirred for 2 h
at 0 °C and then concentrated to dryness. The residue was
dissolved in EtOAc (250 mL) and extracted with saturated
NaHCO3 (50 mL) and brine (50 mL). The organic layer was
dried over MgSO4 and concentrated in vacuo. Flash chroma-
tography (25% hexane/EtOAc) afforded 20 (8.72 g, 64%) as a
355.1303, found 355.1303; [R]24 +31.0 (c 1.10, CHCl3).
D
N-E t h yl-N-[(2R)-2-h yd r oxy-3-(1-p h en ylet h yla m in o)-
p r op yl]tr iflu or om eth a n esu lfon a m id e (r a c-16). Compound
13 (23 mg, 101 µmol) was reacted with (()-1-phenylethylamine
(24 mg, 202 µmol) according to the procedure of r a c-15 and
was purified by flash chromatography (5% CH3OH/CHCl3) to
give r a c-16 (29 mg, 81%) as a colorless oil: 1H NMR (CD3OD)
δ 1.21 (t, 3 H, J ) 7.3), 1.22 (t, 3 H, J ) 7.1), 1.37 (d, 3 H, J
) 6.8), 1.37 (d, 3 H, J ) 6.8), 2.34 (dd, 1 H, J ) 12.3, 8.1), 2.45
(d, 2 H, J ) 6.2), 2.56 (dd, 1 H, J ) 12.3, 4.0), 3.15-3.65 (m,
4 H), 3.75 (q, 1 H, J ) 6.6), 3.76 (q, 1 H, J ) 6.6), 3.85 (m, 1
H), 3.92 (m, 1 H), 7.15-7.35 (m, 10 H).
(2R,10R)-N1,N11-Diet h yl-2,10-d ih yd r oxy-N1,N4,N8,N11
-
1
white solid: mp 64-66 °C; H NMR (CDCl3) δ 1.12 (t, 3 H, J
tetr a k is(ben zyloxyca r bon yl)n or sp er m in e (17a ). Potas-
sium bicarbonate (197 mg, 1.97 mmol) and N-(benzyloxycar-
bonyloxy)succinimide (112 mg, 0.45 mmol) were added to a
mixture of 8a (32 mg, 75 µmol) in H2O (5 mL) and Et2O (5
mL) at 0 °C, and the reaction mixture was stirred at room
temperature for 4 h. Water (20 mL) was added, and the layers
were separated. The aqueous layer was extracted with Et2O
(3 × 30 mL). The combined organic layers were dried over
MgSO4 and concentrated in vacuo. The crude product was
purified by flash chromatography (50% then 60% EtOAc/
cyclohexane) to give 17a (33 mg, 54%) as a colorless oil: 1H
NMR (CD3OD) δ 0.92-1.22 (m, 6 H), 1.55-1.95 (m, 2 H), 2.80-
3.60 (m, 16 H), 3.85-4.10 (m, 2 H), 4.90-5.25 (m, 8 H), 7.10-
7.50 (m, 20 H); HRMS m/z calcd for C45H57N4O10 813.4075,
) 7.3), 2.61 (dd, 1 H, J ) 15.2, 6.8), 2.72 (dd, 1 H, J ) 15.4,
4.0), 3.27 (m, 2 H), 3.73 (d, 1 H, J ) 4.2), 4.53 (m, 1 H), 5.21
(d, 1 H, J ) 12.1), 5.26 (d, 1 H, J ) 12.1), 5.76 (br m, 1 H),
7.32-7.42 (m, 5 H); [R]22D -23.4 (c 1.14, CH3OH). Anal. (C13H17
NO4) C, H, N.
-
(2S)-4-[N-(Ben zyloxyca r bon yl)eth yla m in o]-1,2-bu ta n e-
d iol (21). A solution of BH3 in THF (1 M, 160 mL, 160 mmol)
was added dropwise to 20 (8.50 g, 33.8 mmol) in THF (150
mL) at room temperature under an Ar atmosphere. After 17
h, 4 N HCl was slowly added until the pH reached 4. The
reaction mixture was stirred at room temperature for 4 h, then
concentrated, and neutralized with 1 N NaOH; Et2O (200 mL)
was added. N-(Benzyloxycarbonyloxy)succinimide (9.26 g, 37.2
mmol) and NaHCO3 (2.84 g, 33.8 mmol) were added at 0 °C,
and the mixture was stirred at room temperature for 16 h.
The layers were separated, and the aqueous layer was further
extracted with EtOAc (3 × 100 mL). The organic layers were
washed with NaHCO3 (50 mL), dried over Na2SO4, and solvent
was removed in vacuo. Flash chromatography (5% CH3OH/
CHCl3) gave 21 (3.00 g, 33%) as a colorless oil: 1H NMR (CD3-
OD) δ 1.13 (t, 3 H, J ) 7.0), 1.56 (m, 1 H), 1.80 (m, 1 H), 3.30-
3.55 (m, 6 H), 3.57 (m, 1 H), 5.11 (s, 2 H), 7.25-7.48 (m, 5 H);
13C NMR (CD3OD) δ 13.53, 14.17, 33.07, 33.65, 43.25, 43.60,
44.95, 45.31, 67.32, 68.13, 70.96, 128.83, 129.05, 129.53,
138.25, 157.95; HRMS m/z calcd for C14H22NO4 268.1549, found
268.1542; [R]24D -3.8 (c 1.03, CHCl3). Anal. (C14H21NO4) C, H,
N.
found 813.4064; [R]24 -5.1 (c 1.00, 1 N HCl).
D
(2R,10R)-2,10-Bis[(S)-r-m et h oxy-r-(t r iflu or om et h yl)-
p h en yla cetoxy]-N1,N11-d ieth yl-N1,N4,N8,N11-tetr a k is(ben -
zyloxyca r bon yl)n or sp er m in e (18a ). The reaction was car-
ried out in an oven-dried 5- × 175-mm NMR tube, fitted with
a rubber septum, under an Ar atmosphere. The reagents were
injected via syringe in the following order: anhydrous pyridine
(300 µL), (R)-(-)-Mosher’s acid chloride (24 µL, 108 µmol), CCl4
(200 µL), and then a solution of 17a (23 mg, 28 µmol) in CCl4
(500 µL). The reaction mixture was shaken and allowed to
stand at room temperature for 18 h. CHCl3 (20 mL) was added,
and the solution was washed with saturated NaHCO3 and
brine. The organic layer was dried over MgSO4 and concen-
trated in vacuo. The residue was purified by flash chromatog-
raphy (25% EtOAc/cyclohexane) to give 18a (35 mg, 100%) as
a colorless oil: 1H NMR (CDCl3, 45 °C) δ 1.02 (m, 6 H), 1.50
(m, 2 H), 2.75 (m, 2 H), 3.44 (s, 3 H), 2.85-3.70 (m, 14 H),
4.80-5.20 (m, 8 H), 5.43 (m, 2 H), 7.20-7.48 (m, 30 H); 19F
NMR (CDCl3, CFCl3, 45 °C) δ -71.75; 19F NMR (12% CDCl3/
(2S)-4-[N-(Ben zyloxyca r b on yl)et h yla m in o]-1-p -t olu -
en esu lfon a to-2-bu ta n ol (22). p-TsCl (2.20 g, 11.5 mmol) was
added to a solution of 21 (2.80 g, 10.5 mmol) in CH2Cl2 (20
mL) and pyridine (20 mL) at 0 °C under an Ar atmosphere.
The reaction mixture was stirred for 1 h, then kept at 5 °C for

Hydroxylated Polyamines as Antiproliferatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 233
16 h, and stirred again at 0 °C for 1.5 h. The solution was
diluted with H2O (100 mL) and extracted with CHCl3 (3 × 100
mL). The combined organic layers were washed with 0.5 N
HCl (100 mL) and H2O (100 mL), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash
chromatography (40% EtOAc/hexane) to give 22 (3.16 g, 72%)
as a colorless oil: 1H NMR (CD3OD) δ 1.09 (t, 3 H, J ) 7.0),
1.54 (m, 1 H), 1.69 (m, 1 H), 2.45 (s, 3 H), 3.20-3.40 (m, 4 H),
3.70 (m, 1 H), 3.90 (m, 2 H), 5.10 (s, 2 H), 7.24-7.40 (m, 5 H),
2.42 (s, 3 H), 2.44 (s, 3 H), 3.69-3.77 (m, 1 H), 3.94-4.19 (m,
4 H), 4.30 (d, 1 H, J ) 11.4), 4.49 (d, 1 H, J ) 11.4), 7.12-7.18
(m, 2 H), 7.25-7.36 (m, 7 H), 7.72-7.79 (m, 4 H); [R]24546 +34.1
(lit. en t-33 [R]25 -34.9)39 (c 0.64, CHCl3).
546
(2R)-2-(Ben zyloxy)-4-[N-(m esitylen esu lfon yl)eth yla m i-
n o]-O-tosyl-1-bu ta n ol (34). Sodium hydride (60%, 1.5 g, 37
mmol) was added to N-ethylmesitylenesulfonamide42 (6.5 g,
29 mmol) in DMF (20 mL) at 0 °C under a N2 atmosphere,
and the mixture was stirred for 0.5 h. A solution of 33 (18.7 g,
37.1 mmol) in DMF (100 mL) was added to the reaction
mixture, followed by stirring for 16 h at room temperature.
The mixture was cooled in an ice bath, cautiously quenched
with H2O (10 mL), concentrated in vacuo, and extracted with
EtOAc (150 mL). The extract was washed with H2O (150 mL)
and brine (150 mL) and then dried over Na2SO4. Solvent
removal in vacuo followed by flash chromatography (25%
EtOAc/cyclohexane) gave 34 (19.8 g, 77%) as a colorless oil:
1H NMR (CDCl3) δ 1.01 (t, 3 H, J ) 7.1), 1.69 (m, 2 H), 2.27
(s, 3 H), 2.44 (s, 3 H), 2.55 (s, 6 H), 3.11-3.32 (m, 4 H), 3.51-
3.59 (m, 1 H), 3.94 (d, 1 H, J ) 4.8), 3.96 (d, 1 H, J ) 4.8),
4.37 (d, 1 H, J ) 11.4), 4.41 (d, 1 H, J ) 11.4), 6.90 (s, 2 H),
7.17-7.21 (m, 2 H), 7.28-7.34 (m. 5 H), 7.73-7.78 (m, 2 H);
13C NMR (CDCl3) δ 12.79, 20.87, 21.60, 22.69, 29.61, 40.33,
41.38, 70.60, 71.99, 74.06, 127.67, 127.74, 127.87, 128.30,
129.87, 131.90, 132.70, 133.19, 137.65, 140.03, 142.34, 144.92;
HRMS calcd for C29H38NO6S2 560.2141 (M + 1), found 560.2124;
7.42 (d, 2 H, J ) 8.1), 7.78 (m, 2 H); HRMS m/z calcd for C21H28
-
NO6S 422.1637, found 422.1664; [R]22 -0.8 (c 0.98, CHCl3).
D
Anal. (C21H27NO6S) C, H, N.
(3S )-N -(Be n zyloxyca r b on yl)-N -e t h yl-3,4-e p oxyb u t -
yla m in e (23). Potassium Bicarbonate (1.13 g, 8.18 mmol) in
CH3OH (100 mL) was added to 22 (3.13 g, 7.44 mmol). The
mixture was stirred at room temperature under an Ar atmo-
sphere for 100 min, then poured into a mixture of H2O (100
mL) and CH2Cl2 (100 mL). The layers were separated, and
the aqueous layer was extracted with CH2Cl2 (3 × 100 mL).
The combined organic extracts were dried over Na2SO4, and
the solvent was removed in vacuo. Flash chromatography (33%
EtOAc/hexane) afforded 23 (1.59 g, 86%) as a colorless oil: 1H
NMR (CDCl3) δ 1.14 (t, 3 H, J ) 6.8), 1.78 (m, 1 H), 1.86 (m,
1 H), 2.45 (m, 1 H), 2.72 (m, 1 H), 2.92 (m, 1 H), 3.24-3.55
(m, 4 H), 5.14 (s, 2 H), 7.25-7.44 (m, 5 H); 13C NMR (CDCl3)
δ 13.25, 13.87, 31.56, 32.07, 42.12, 42.46, 43.52, 44.28, 46.89,
50.14, 66.89, 127.77, 127.86, 128.41, 136.85, 155.94; HRMS
m/z calcd for C14H20NO3 250.1443, found 250.1442; [R]23D -11.6
(c 0.97, CHCl3). Anal. (C14H19NO3) C, H, N.
[R]23 +14 (c 1.00, CH3OH). Anal. (C29H37NO6S2) C, H, N.
D
(3R,12R)-Diben zyloxy-N1,N14-d ieth yl-N1,N5,N10,N14-tet-
r a k is(m esitylen esu lfon yl)h om osp er m in e (35). Sodium hy-
dride (60%, 1.37 g, 33.9 mmol) was added to N,N′-bis-
(mesitylenesulfonyl)-1,4-butanediamine40 (5.1 g, 11 mmol) in
DMF (50 mL) at 0 °C under a N2 atmosphere, and the mixture
was stirred for 0.5 h. Sodium iodide (107 mg, 0.70 mmol) and
34 (13.3 g, 23.6 mmol) in DMF (100 mL) were added, followed
by stirring at 60 °C for 16 h. The mixture was cooled in an ice
bath, cautiously quenched with H2O (20 mL), concentrated in
vacuo, and extracted with EtOAc (200 mL). The extract was
washed with 1% NaHSO3 (150 mL), H2O (2 × 150 mL), and
brine (150 mL), then dried over Na2SO4. Solvent removal in
vacuo followed by flash chromatography (1:2:7 MeOAc:petro-
leum ether:toluene) gave 35 (7.0 g, 50%) as a colorless oil: 1H
NMR (CDCl3) δ 0.98 (t, 6 H, J ) 7.2) 1.46-1.65 (m, 8 H), 2.26
(s, 6 H), 2.28 (s, 6 H), 2.54 (s, 12 H), 2.55 (s, 12 H), 3.04-3.18
(m, 16 H), 3.28-3.36 (m, 2 H), 4.24 (d, 2 H, J ) 11.4), 4.32 (d,
2 H, J ) 11.4), 6.90 (d, 8 H, J ) 5.7), 7.18-7.34 (m, 10 H);
HRMS calcd for C66H91N4O10S4 1227.5618 (M + 1), found
(3S,12S)-N1,N14-Diet h yl-3,12-d ih yd r oxy-N1,N5,N10,N14
-
tetr a k is(ben zyloxyca r bon yl)h om osp er m in e (28a ). A solu-
tion of 23 (1.18 g, 4.33 mmol) and 24 (209 mg, 2.37 mmol) was
heated in EtOH (100 mL) at 55 °C for 3 days under an Ar
atmosphere. The solvent was removed under reduced pressure
to give 26, which was dissolved in CHCl3 (100 mL) and cooled
to 0 °C, followed by addition of benzyl chloroformate (1.01 g,
5.93 mmol) and triethylamine (959 mg, 9.48 mmol). The ice
bath was removed after 5 min, and the reaction mixture was
stirred at room temperature for 2 h and diluted with CHCl3
(100 mL), which was washed with 1 N HCl (20 mL), H2O (20
mL), and brine (20 mL). The organic layer was dried over
MgSO4 and concentrated in vacuo. Purification by flash
chromatography (33%, then 25% hexane/EtOAc, then 10%
CH3OH/CHCl3) gave 28a (453 mg, 22%) as a colorless oil: 1H
NMR (CD3OD) δ 1.10 (m, 6 H), 1.35-1.82 (m, 8 H), 2.90-3.55
(m, 16 H), 3.74 (m, 2 H), 5.09 (s, 8 H), 7.22-7.42 (m, 20 H);
HRMS m/z calcd for C18H63N4O10 855.4544, found 855.4500;
1227.5701; [R]23 +0.8 (c 1.00, CH3OH).
[R]21 +3.3 (c 0.97, CH3OH).
D
D
(3R,12R)-N1,N14-Dieth yl-3,12-d ih yd r oxy-N1,N5,N10,N14
-
(3S ,12S )-N 1,N 14-D ie t h y l-3,12-d ih y d r o x y h o m o s p e r -
m in e Tetr a h yd r och lor id e (29a ). Hydrochloric acid (1 N, 4.0
mL) and 10% Pd-C (50 mg) were introduced into 28a (427
mg, 0.50 mmol) in EtOH (40 mL). The reaction mixture was
stirred under H2 (1 atm) for 20 h, filtered through Celite, and
concentrated in vacuo. The residue was taken up in H2O (3 ×
5 mL), filtered through a syringe filter (0.2 µm), and concen-
trated in vacuo. Recrystallization from aqueous EtOH gave
29a (196 mg, 85%) as white crystals: 1H NMR (D2O, NaTSP)
δ 1.30 (t, 6 H, J ) 7.3), 1.74-2.04 (m, 8 H), 2.98-3.32 (m, 16
H), 4.05 (tt, 2 H, J ) 9.6, 3.3); 13C NMR (D2O, 1,4-dioxane )
67.19 ppm) δ 11.14, 23.25, 31.00, 43.63, 44.39, 47.58, 52.70,
tetr a k is(m esitylen esu lfon yl)h om osp er m in e (36). A solu-
tion of 35 (9.33 g, 7.60 mmol) in HOAc (300 mL) and H2O (15
mL) was divided into two about equal portions. Palladium
black was added to each (1.03 and 1.07 g), and the mixtures
were shaken under 1 atm H2 at room temperature (3.5 and 4
h, respectively).41 The mixtures were filtered through Celite;
solids were washed with HOAc (3 × 50 mL). Combined
washings and filtrates were concentrated in vacuo; the result-
ing oil was purified by flash chromatography (50% hexane/
EtOAc) to give 36 (6.87 g, 86%) as a white foam: mp 135-
1
137 °C; H NMR (CDCl3) δ 0.98 (t, 6 H, J ) 7.5), 1.33-1.54
(m, 8 H), 2.29 (s, 6 H), 2.30 (s, 6 H), 2.57 (s, 24 H), 3.02-3.32
(m, 14 H), 3.36-3.48 (m, 2 H), 3.69-3.81 (br m, 2 H), 6.94 (s,
4 H), 6.95 (s, 4 H); HRMS calcd for C52H79N4O10S4 1047.4679
65.22; HRMS m/z calcd for
C16H39N4O2 319.3073, found
319.3073; [R]23 +8.7 (c 1.05, 1 N HCl). Anal. (C16H42Cl4N4O2)
D
C, H, Cl, N.
(M + 1), found 1047.4710; [R]26 -2.4 (c 0.97, CHCl3). Anal.
D
Dim eth yl (2R)-2-(Ben zyloxy)su ccin a te (31). Compound
31 was prepared by benzylation of (R)-dimethyl malate with
benzyl 2,2,2-trichloroacetimidate in 85% yield using a litera-
ture method:36 [R]24 +70.7 (lit. [R]23 +70)36 (c 1.0, CHCl3).
(C52H78N4O10S4) C, H, N.
(3R ,12R )-N 1,N 14-Die t h yl-3,12-d ih yd r oxyh om osp e r -
m in e Tetr a h yd r och lor id e (29b). A solution of 36 in DME
(80 mL) was cooled to -78 °C under a N2 atmosphere and
titrated with a solution of sodium naphthalenide in DME
[freshly prepared by stirring Na (3.00 g, 130 mmol) and
naphthalene (19.50 g, 143 mmol) in DME (80 mL) under N2
for 2.5 h at room temperature].23 Upon reaching a dark green
endpoint, the mixture was quenched with saturated NaHCO3
(25 mL) and allowed to warm to room temperature. Potassium
carbonate (45 g) was added, and the mixture was stirred
D
D
(2R)-2-(Ben zyloxy)-1,4-bu ta n ed iol (32). Compound 32
was made in 83% yield using a literature procedure37 by
reduction of 31 modified with the use of LiBH4: [R]23 +15
D
(lit. [R]23 +15)37 (c 1.0, CHCl3).
D
(2R)-2-(Ben zyloxy)-O,O′-ditosyl-1,4-bu tan ediol (33). Com-
pound 33 was made in 87% yield by treatment of 32 with
p-TsCl in pyridine by a known method:38 mp 86-87 °C (lit.
1
en t-33)39 mp 93 °C; H NMR (CDCl3) δ 1.72-1.91 (m, 2 H),

234 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Bergeron et al.
overnight under N2. Diethyl ether (150 mL) was added, the
mixture was filtered, and solids were washed with Et2O (3 ×
50 mL). The combined filtrate and washings were concentrated
in vacuo, and 1 N HCl (130 mL) was added. The mixture was
acidified to pH 1 with concentrated HCl, then filtered; solvents
were removed in vacuo. The residue was suspended in EtOH
(160 mL) and centrifuged; the brown supernatant was de-
canted. The remaining solids were dissolved in 2 N NaOH (15
mL) and extracted with Et2O (10 × 100 mL). The combined
extracts were dried over Na2SO4 and filtered, and solvents
were removed in vacuo. The product was dissolved in EtOH
(40 mL) and acidified to pH 1 with 1 N HCl. Solvent removal
in vacuo gave 0.35 g of 29b (21%) as white plates: mp > 280
°C; 1H NMR (D2O, NaTSP) δ 1.29 (t, 6 H, J ) 7.4), 1.75-2.03
(m, 8 H), 3.02-3.38 (m, 16 H), 4.05 (tt, 2 H, J ) 9.7, 3.2); 13C
NMR (D2O, 1,4-dioxane ) 67.19 ppm) δ 11.16, 23.24, 31.00,
43.64, 44.41, 47.57, 52.69, 65.22; HRMS calcd for C16H39N4O2
319.3073 (M + 1), found 319.3102; [R]25D -8.6 (c 1.00, 1 N HCl).
Anal. (C16H42Cl4N4O2) C, H, N.
% of control growth )
final treated cell no. - initial inoculum
final untreated cell no. - initial inoculum
× 100
The IC50 is defined as the concentration of compound necessary
to reduce cell growth to 50% of control growth after defined
intervals of exposure.
P olya m in e P ool An a lysis. While in logarithmic growth,
cells were treated with the polyamine derivatives. At the end
of the treatment period, cell suspensions were sampled,
washed twice in ice-cold, incomplete medium, and pelleted for
extraction using 0.6 N perchloric acid,40 then freeze-fractured
in liquid nitrogen/hot water three times. Each supernatant was
frozen at -20 °C until analysis of polyamine content by
HPLC.19
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 cells in vitro.5,40 Cell suspensions were
incubated in 1 mL of culture medium containing 1, 2, 4, 6, 8,
and 10 µM radiolabeled SPD alone or in the presence of 10,
25, and 50 µM polyamine analogue for 20 min at 37 °C. At
the end of the incubation period, tubes were centrifuged at
1200 rpm for 5 min at 0-4 °C. The pellet was washed twice
with 5 mL of cold RPMI-1640 containing 1 mM SPD, digested
in 200 µL of 1 N NaOH at 60 °C for 1 h, and neutralized with
500 µL 1 N HCl. The material was transferred to a vial for
scintillation counting. Lineweaver-Burke 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 Pegg43
and Pegg and Po¨so¨,44 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.
Spermidine/spermine N1-acetyltransferase activity was based
on quantitation of [14C]-N1-acetylspermidine formed by acety-
lation of SPD with [14C]acetyl coenzyme A according to the
method of Libby et al.11 Cells treated with DENSPM were
positive controls.
(3S,12S)-3,12-Bis[(S)-r-m et h oxy-r-(t r iflu or om et h yl)-
ph en ylacetoxy]-N1,N14-dieth yl-N1,N5,N10,N14-tetr akis(ben -
zyloxyca r bon yl)h om osp er m in e (37a ). Compound 28a (20
mg, 23 µmol) was reacted with (R)-(-)-Mosher’s acid chloride
(14 µL, 73 µmol) using the procedure of 18a . Flash chroma-
tography (33% EtOAc/hexane) furnished 37a (27 mg, 90%) as
a colorless oil: 1H NMR (CDCl3, 45 °C) δ 1.02 (t, 6 H, J ) 7.0),
1.32 (m, 4 H), 1.82 (m, 4 H), 2.90-3.54 (m, 16 H), 3.43 (s, 6
H), 5.09 (s, 8 H), 5.23 (m, 2 H), 7.20-7.60 (m, 30 H); 19F NMR
(CDCl3, CFCl3 as internal standard, 45 °C) δ -71.57; HRMS
m/z calcd for C68H77F6N4O14 1287.5340, found 1287.5319; [R]22
D
-7.7 (c 1.35, CHCl3).
(3R,12R)-N1,N14-Dieth yl-3,12-d ih yd r oxy-N1,N5,N10,N14
-
t et r a k is(b en zyloxyca r b on yl)h om osp er m in e (28b ).
A
sample of 29b (50 mg, 108 µmol) was reacted with N-(benzyl-
oxycarbonyloxy)succinimide (162 mg, 0.65 mmol) utilizing the
procedure of 17a . Purification by flash chromatography (25%
hexane/EtOAc) gave 28b (59 mg, 64%) as a colorless oil: 1H
NMR (CD3OD) δ 1.10 (m, 6 H), 1.35-1.82 (m, 8 H), 2.94-3.50
(m, 16 H), 3.74 (m, 2 H), 5.09 (s, 8 H), 7.22-7.38 (m, 20 H);
HRMS m/z calcd for C48H63N4O10 855.4544, found 855.4513;
[R]25 -3.2 (c 0.95, CH3OH).
D
(3R,12R)-3,12-Bis[(S)-r-m et h oxy-r-(t r iflu or om et h yl)-
ph en ylacetoxy]-N1,N14-dieth yl-N1,N5,N10,N14-tetr akis(ben -
zyloxyca r bon yl)h om osp er m in e (37b). Compound 28b (36
mg, 42 µmol) was reacted with (R)-(-)-Mosher’s acid chloride
(30 µL, 161 µmol) as in the preparation of 18a . Flash
chromatography (33% EtOAc/hexane) gave 37b (50 mg, 92%)
as a colorless oil: 1H NMR (CDCl3, 45 °C) δ 1.05 (t, 6 H, J )
6.6), 1.12-1.36 (m, 4 H), 1.72-1.98 (m, 4 H), 2.70-2.88 (m, 2
H), 2.90-3.09 (m, 2 H), 3.11-3.42 (m, 12 H), 3.48 (s, 6 H),
4.97-5.08 (m, 4 H), 5.11 (s, 2 H), 5.17-5.33 (m, 4 H), 7.23-
7.52 (m, 30 H); 19F NMR (CDCl3, CFCl3 as internal standard,
45 °C) δ -71.80; HRMS m/z calcd for C68H77F6N4O14 1287.5340,
Ack n ow led gm en t. The authors thank SunPharm
Corp., Ponte Vedra Beach, FL, for financial support. We
appreciate Curt O. Zimmerman and Samuel E. Algee
for their technical assistance and Dr. Eileen Eiler-
Hughes for her editorial comments.
Refer en ces
(1) Bergeron, R. J .; Neims, A. H.; McManis, J . S.; Hawthorne, T.
R.; Vinson, J . R. T.; Bortell, R.; Ingeno, M. J . Synthetic
Polyamine Analogues as Antineoplastics. J . Med. Chem. 1988,
31, 1183-1190.
(2) Bernacki, R. J .; Bergeron, R. J .; Porter, C. W. Antitumor Activity
of N,N′-Bis(ethyl)spermine Homologues against Human
MALME-3 Melanoma Xenografts. Cancer Res. 1992, 52, 2424-
2430.
(3) Porter, C. W.; Bergeron, R. J .; Stolowich, N. J . Biological
Properties of N4-Spermidine Derivatives and Their Potential in
Anticancer Chemotherapy. Cancer Res. 1982, 42, 4072-4078.
(4) Porter, C. W.; Miller, J .; Bergeron, R. J . Aliphatic Chain Length
Specificity of the Polyamine Transport System in Ascites L1210
Leukemia Cells. Cancer Res. 1984, 44, 126-128.
(5) Bergeron, R. J .; Hawthorne, T. R.; Vinson, J . R. T.; Beck, D. E.,
J r.; Ingeno, M. J . Role of the Methylene Backbone in the
Antiproliferative Activity of Polyamine Analogues on L1210
Cells. Cancer Res. 1989, 49, 2959-2964.
(6) Pegg, A. E.; Madhubala, R.; Kameji, T.; Bergeron, R. J . Control
of Ornithine Decarboxylase Activity in R-Difluoromethylorni-
thine-resistant L1210 Cells by Polyamines and Synthetic Ana-
logues. J . Biol. Chem. 1988, 263, 11008-11014.
(7) Porter, C. W.; Pegg, A. E.; Ganis, B.; Madhabala, R.; Bergeron,
R. J . Combined Regulation of Ornithine and S-Adenosylme-
thionine Decarboxylases by Spermine and the Spermine Ana-
logue N1,N12-Bis(ethyl)spermine. Biochem. J . 1990, 268, 207-
212.
found 1287.5384; [R]25 -22.4 (c 1.33, CHCl3).
D
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-
dine (Sigma) at 37 °C in a water-jacketed 5% CO2 incubator.
IC50 Deter m in a tion s. Cells were grown in 25-cm2 tissue
culture flasks in a total volume of 10 mL. Cultures were
treated while in logarithmic growth (0.5-1.0 × 105 cells/mL)
with the polyamine derivatives dissolved in sterile water and
filtered through a 0.2-µm syringe filter. Following a 48-h
period, cells were reseeded and incubated for an additional
48 h.
After the indicated time periods, 200 µL of cell suspension
was removed from the flask for counting. Cell number was
determined by electronic particle counting (model ZF coulter
counter, Coulter Electronics, Hialeah, FL) and confirmed
periodically with hemocytometer measurements.
The percentage of control growth was determined as follows:

Hydroxylated Polyamines as Antiproliferatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 235
(8) Porter, C. W.; McManis, J .; Casero, R. A.; Bergeron, R. J .
Relative Abilities of Bis(ethyl) Derivatives of Putrescine, Sper-
midine, and Spermine to Regulate Polyamine Biosynthesis and
Inhibit L1210 Leukemia Cell Growth. Cancer Res. 1987, 47,
2821-2825.
(9) Pegg, A. E.; Wechter, R.; Pakala, R.; Bergeron, R. J . Effect of
N1,N12-Bis(ethyl)Spermine and Related Compounds on Growth
and Polyamine Acetylation, Content, and Excretion in Human
Colon Tumor Cell. J . Biol. Chem. 1989, 264, 11744-11749.
(10) Libby, P. R.; Henderson, M.; Bergeron, R. J .; Porter, C. W. Major
Increases in Spermidine/Spermine-N1-Acetyltransferase Activity
by Spermine Analogues and Their Relationship to Polyamine
Depletion and Growth Inhibition in L1210 Cells. Cancer Res.
1989, 49, 6226-6231.
(11) Libby, P. R.; Bergeron, R. J .; Porter, C. W. Structure-Function
Correlations of Polyamine Analog-Induced Increases in Sper-
midine/Spermine Acetyltransferase Activity. Biochem. Pharma-
col. 1989, 38, 1435-1442.
(12) Casero, R. A., J r.; Celano, P.; Ervin, S. J .; Porter, C. W.;
Bergeron, R. J .; Libby, P. Differential Induction of Spermidine/
Spermine N1-Acetyltransferase in Human Lung Cancer Cells by
the Bis(ethyl)Polyamine Analogues. Proc. Am. Assoc. Cancer Res.
1989, 49, 3829-3833.
(13) Porter, C. W.; Ganis, B.; Libby, P. R.; Bergeron, R. J . Correlations
between Polyamine Analogue-Induced Increases in Spermidine/
Spermine N1-Acetyltransferase Activity, Polyamine Pool Deple-
tion, and Growth Inhibition in Human Melanoma Cell Lines.
Cancer Res. 1991, 51, 3715-3720.
(14) Bergeron, R. J .; Feng, Y.; Weimar, W. R.; McManis, J . S.;
Dimova, H.; Porter, C.; Raisler, B.; Phanstiel, O. A Comparison
of Structure-Activity Relationships between Spermidine and
Spermine Analogue Antineoplastics. J . Med. Chem. 1997, 40,
1475-1494.
(15) Fogel-Petrovic, M.; Shappell, N. W.; Bergeron, R. J .; Porter, C.
W. Polyamine and Polyamine Analogue Regulation of Spermi-
dine/Spermine N1-Acetyltransferase in MALME-3M Human
Melanoma Cells. J . Biol. Chem. 1993, 268, 19118-19125.
(16) Shappell, N. W.; Fogel-Petrovic, M. F.; Porter, C. W. Regulation
of Spermidine/Spermine N1-Acetyltransferase by Intracellular
Polyamine Pools - Evidence for a Functional Role in Polyamine
Homeostasis. FEBS Lett. 1993, 321, 179-183.
(17) Bergeron, R. J .; Yao, G. W.; Yao, H.; Weimar, W. R.; Sninsky,
C. A.; Raisler, B.; Feng, Y.; Wu, Q.; Gao, F. Metabolically
Programmed Polyamine Analogue Antidiarrheals. J . Med. Chem.
1996, 39, 2461-2471.
(18) Bergeron, R. J .; Wiegand, J .; Sninsky, C. A.; Katovich, M. J . The
Impact of Polyamine Analogues on the Blood Pressure of
Normotensive and Hypertensive Rats. Clin. Exp. Hypertension
1995, 17, 1197-1217.
(19) Bergeron, R. J .; Weimar, W. R.; Luchetta, G.; Streiff, R. R.;
Wiegand, J .; Perrin, J .; Schreier, K. M.; Porter, C.; Yao, G. W.;
Dimova, H. Metabolism and Pharmacokinetics of N1,N11-Dieth-
ylnorspermine. Drug Metab. Dispos. 1995, 23, 1117-1125.
(20) Bergeron, R. J .; Weimar, W. R.; Luchetta, G.; Sninsky, C. A.;
Wiegand, J . Metabolism and Pharmacokinetics of N1,N14-Dieth-
ylhomospermine. Drug Metab. Dispos. 1996, 24, 334-343.
(21) Niitsu, M.; Samejima, K. Syntheses of a Series of Linear
Polyamines with Three and Four Methylene Chain Intervals.
Chem. Pharm. Bull. 1986, 34, 1032-1038.
(22) Baldwin, J . J .; Raab, A. W.; Mensler, K.; Arison, B. H.; McClure,
D. E. Synthesis of (R)- and (S)-Epichlorohydrin. J . Org. Chem.
1978, 43, 4876-4878.
(23) Heathcock, C. H.; Blumenkopf, T. A.; Smith, K. M. Total
Synthesis of (()-Fawcettime. J . Org. Chem. 1989, 54, 1548-
1562.
(24) Henry, J . R.; Marcin, L. R.; McIntosh, M. C.; Scola, P. M.; Harris,
G. D., J r. Mitsunobu Reactions of N-Alkyl and N-Acyl Sulfona-
mides - An Efficient Route to Protected Amines. Tetrahedron
Lett. 1989, 30, 5709-5712.
(25) Edwards, M. L.; Stemerick, D. M.; McCarthy, J . R. Use of the
Mitsunobu Reaction in the Synthesis of Polyamines. Tetrahedron
1994, 50, 5579-5590.
(26) Lohray, B. B.; Reddy, A. S.; Bhushan, V. An Efficient Synthesis
of (3R)-4-Amino-3-hydroxy Butyric Acid (GABOB) via Cyclic
Sulfite Methodology. Tetrahedron: Asymmetry 1996, 7, 2411-
2416.
(27) The less acidic ethyltosylamide
1 reacted with (R)-(-)-2,2-
dimethyl-1,3-dioxolane-4-methanol in a less satisfactory yield of
43%.
(28) Hendrickson, J . B.; Bergeron, R. J . Triflamides for Protection
and Monoalkylation of Amines and a New Gabriel Synthesis.
Tetrahedron Lett. 1973, 39, 3839-3842.
(29) Dale, J . A.; Mosher, H. S. Nuclear Magnetic Resonance Enan-
tiomer Reagents. Configurational Correlations via Nuclear
Magnetic Resonance Chemical Shifts of Diastereomeric Man-
delate, O-Methylmandelate, and R-Methoxy-R-trifluorometh-
ylphenylacetate (MTPA) Esters. J . Am. Chem. Soc. 1973, 95,
512-519.
(30) Mixing experiments showed that a 7% contaminant of 18b is
easily observable in the 19F NMR spectrum (282 MHz, 12%
CDCl3/CD3OD, at 45 °C) of 18a .
(31) Partial racemization in the epoxide synthons (5, 13, or 23) or
the alternate synthon (34) would lead to meso-tetraamines as
the main contaminants. Since the stereocenters in the hydroxy-
lated polyamines are far apart, the Mosher’s esters of the
respective CBZ-protected enantiomers were employed as model
compounds.
(32) Bergeron, R. J .; Weimar, W. R.; Mu¨ller, R.; Zimmerman, C. O.;
McCosar, B. H.; Yao, H.; Smith, R. E. Synthesis of Reagents for
the Construction of Hypusine and Deoxyhypusine Peptides and
their Application as Peptidic Antigens. J . Med. Chem. 1998, 41,
3888-3900.
(33) Danielmeier, K.; Steckhan, E. Efficient Pathways to (R)-and (S)-
5-Hydroxymethyl-2-oxazolidinone and Some Derivatives. Tet-
rahedron: Asymmetry 1995, 6, 1181-1190.
(34) Cammas, S.; Renard, I.; Boutault, K.; Gue´rin, P. A Novel
Synthesis of Optically Active 4-Benzyloxy- and 4-Alkyloxycar-
bonyl-2-Oxetanones. Tetrahedron: Asymmetry 1993, 4, 1925-
1930.
(35) Miller, M. J .; Bajwa, J . S.; Mattingly, P.; Peterson, K. Enanti-
oselective Syntheses of 3-Substituted 4-(Alkoxycarbonyl)-2-aze-
tidinones from Malic Acid. J . Org. Chem. 1982, 47, 4928-4933.
(36) Widmer, U. A Convenient Benzylation Procedure for â-Hydroxy
Ethers. Synthesis 1987, 568-570.
(37) Gmeiner, P.; J unge, D. Regioselective Transformation of Malic
Acid: A Practical Method for the Construction of Enantiomeri-
cally Pure Indolizidines. J . Org. Chem. 1995, 60, 3910-3915.
(38) Benes, J .; Hetflejs, J . Catalysis by Metal Complexes. XXXIX.
Synthesis of Some Phosphines Containing Chiral Substituents.
Collect. Czech. Chem. Commun. 1976, 41, 2256-2263.
(39) Bergeron, R. J .; McManis, J . S.; Perumal, P. T.; Algee, S. E. The
Total Synthesis of Alcaligin. J . Org. Chem. 1991, 56, 5560-5563.
(40) Bergeron, R. J .; McManis, J . S.; Liu, C. Z.; Feng, Y.; Weimar,
W. R.; Luchetta, G. R.; Wu, Q.; Ortiz-Ocasio, J .; Vinson, J . R.
T.; Kramer, D.; Porter, C. Antiproliferative Properties of
Polyamine Analogues: A Structure-Activity Study. J . Med.
Chem. 1994, 37, 3464-3476.
(41) Felix, A. M.; Heimer, E. P.; Lambros, T. J .; Tzougraki, C.;
Meienhofer, J . Rapid Removal of Protecting Groups from Pep-
tides by Catalytic Transfer Hydrogenation with 1,4-Cyclohexa-
diene. J . Org. Chem. 1978, 43, 4194-4196.
(42) Schreinemakers, F. A. H. Some Amides of Aromatic Sulfonic
Acids. Recl. Trav. Chim. Pays-Bas Belg. 1897, 16, 411-424.
(43) Seely, J . E.; Pegg, A. E. Ornithine Decarboxylase (Mouse
Kidney). Methods Enzymol. 1983, 94, 158-161.
(44) Pegg, A. E.; Po¨so¨, H. S-Adenosylmethionine Decarboxylase (Rat
Liver). Methods Enzymol. 1983, 94, 234-239.
J M990375Z