α-methyl polyamines: Efficient synthesis and tolerance studies in vivo and in vitro. First evidence for dormant stereospecificity of polyamine oxidase

Article abstract of DOI:10.1021/jm050872h

Efficient syntheses of metabolically stable α-methylspermidine 1, α-methylspermine 2, and bis-α,α-methylated spermine 3 starting from ethyl 3-aminobutyrate are described. The biological tolerance for these compounds was tested in wild-type mice and transgenic mice carrying the metallothionein promoter-driven spermidine/spermine N¹- acetyltransferase gene (MT-SSAT). The efficient substitution of natural polyamines by their derivatives was confirmed in vivo with the rats harboring the same MT-SSAT transgene and in vitro with the immortalized fibroblasts derived from these animals. Enantiomers of previously unknown 1-amino-8-acetamido-5-azanonane dihydrochloride 4 were synthesized starting from enantiomerically pure (R)- and (S)-alaninols. The studies with recombinant human polyamine oxidase (PAO) showed that PAO (usually splits achiral substrates) strongly favors the (R)-isomer of 4 that demonstrates for the first time that the enzyme has hidden potency for stereospecificity.

Full text of DOI:10.1021/jm050872h

J. Med. Chem. 2006, 49, 399-406
399
r-Methyl Polyamines: Efficient Synthesis and Tolerance Studies in Vivo and in Vitro. First
Evidence for Dormant Stereospecificity of Polyamine Oxidase
Aki J. Ja¨rvinen,†,‡ Marc Cerrada-Gimenez,†,‡ Nikolay A. Grigorenko,§ Alex R. Khomutov,§ Jouko J. Vepsa¨la¨inen,*,||
Riitta M. Sinervirta,† Tuomo A. Keina¨nen,† Leena I. Alhonen,† and Juhani E. Ja¨nne†
Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, and Department of Chemistry,
UniVersity of Kuopio, Kuopio, Finland, and Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
ReceiVed September 4, 2005
Efficient syntheses of metabolically stable R-methylspermidine 1, R-methylspermine 2, and bis-R,R′-
methylated spermine 3 starting from ethyl 3-aminobutyrate are described. The biological tolerance for these
compounds was tested in wild-type mice and transgenic mice carrying the metallothionein promoter-driven
spermidine/spermine N1-acetyltransferase gene (MT-SSAT). The efficient substitution of natural polyamines
by their derivatives was confirmed in vivo with the rats harboring the same MT-SSAT transgene and in
vitro with the immortalized fibroblasts derived from these animals. Enantiomers of previously unknown
1-amino-8-acetamido-5-azanonane dihydrochloride 4 were synthesized starting from enantiomerically pure
(R)- and (S)-alaninols. The studies with recombinant human polyamine oxidase (PAO) showed that PAO
(usually splits achiral substrates) strongly favors the (R)-isomer of 4 that demonstrates for the first time that
the enzyme has hidden potency for stereospecificity.
Scheme 1. Polyamine Metabolism in Mammalsa
Introduction
Under the physiological conditions, biogenic polyamines are
polycations that interact with many cellular macromolecules and
participate in several distinct functions in the mammalian
physiology.1 Precisely maintained polyamine homeostasis is
imperative for cell proliferation as both the accumulation and
depletion of polyamines can lead to disorders of cellular
metabolism.2 In many tumor cell lines, the polyamine levels
are high compared to normal cells, supporting accelerated cell
proliferation. On the other hand, the depletion of the polyamine
pools with inhibitors of their biosynthesis or compounds
activating the catabolism of the polyamines lead to cell growth
inhibition and even to apoptosis.3
In mammalian cells (Scheme 1) spermine can be oxidized
directly to spermidine by spermine oxidase.4,5 Another pos-
sibility for the back-conversion of spermine to spermidine (and
similarly spermidine to putrescine) is the concatenated actions
of spermidine/spermine N1-acetyltransferase (SSAT) and
polyamine oxidase (PAO). SMO uses only spermine, whereas
PAO strongly favors N1-acetylated substrates; both singly or
doubly acetylated spermine and N1-acetylspermidine are excel-
lent substrates for PAO.6 This back-conversion of the polyamines
produces one H2O2 molecule per each split carbon-nitrogen
bond, thus causing oxidative stress. Drugs affecting polyamine
catabolism may induce SSAT activity up to 1000-fold and cause
the depletion of the higher polyamines spermidine and spermine.
Such compounds are of great interest and are being studied as
anticancer drugs.7
a PAO, polyamine oxidase; SMO, spermine oxidase; SPDSY, spermidine
synthase; SPMSY, spermine synthase; SSAT, spermidine/spermine N1-
acetyltransferase.
diethylnorspermine, DENSpm) or nonsymmetrically bis-alkyl-
ated at terminal amino groups. These compounds, similar to
spermine, are actively transported into the cells but are incapable
of fulfilling crucial cellular functions of polyamines.8 The
accumulation of DENSpm, or other compounds of similar
nature, results in the down-regulation of the polyamine synthesis
and/or the activation of the polyamine catabolism. Such
terminally N-alkylated analogues are substrates for PAO9 and
after terminal dealkylation are prone to the SSAT-PAO
pathway.
A less investigated group of polyamine derivatives comprises
the analogues with an alkyl substituent(s) attached to carbon
atom(s) of polyamine backbone. Contrary to terminally N-
alkylated derivatives of spermine, some of these compounds
are able to sustain cell viability with depleted polyamine
pools.10,11 The single methyl group in the R-position (Figure 1)
prevents the acetylation of the adjacent amine of R-methylsper-
midine (1, R-MeSPD), R-methylspermine (2, R-MeSPM), and
bis-R,R′-methylated spermine (3, R,R′-Me2SPM) by SSAT,
Alkylated polyamine derivatives are widely used for the
investigation of the biochemistry of spermine and spermidine.
Most of these studies have concentrated on the analogues of
spermine and its homologues that are symmetrically (e.g.
* To whom the correspondence should be addressed. Tel: +358-17-
163256. Fax: +358-17-163259. E-mail: Jouko.Vepsalainen@uku.fi.
† Department of Biotechnology and Molecular Medicine, University of
Kuopio.
‡ These authors contributed equally to the work.
§ Russian Academy of Sciences.
|| Department of Chemistry, University of Kuopio.
10.1021/jm050872h CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/15/2005

400 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Ja¨rVinen et al.
the case of SSAT overinduction, exogenous supplementation
of the natural polyamines does not restore cell viability due to
their rapid intracellular catabolism.19 However, treatment with
1 prevented the acute pancreatitis altogether in vivo18 and both
1 and 3 restored the early liver regeneration in the partially
hepatectomized transgenic rats.17,18 Moreover, we showed that
1 and 3 are competitive SSAT inhibitors and rather poor
substrates for the oxidases (especially 1) involved in the
catabolism of the polyamines in vitro.17
Here we present efficient synthetic protocols to prepare 1-3
in quantities sufficient for animal studies. Synthesis of previously
unknown 1-amino-8-acetamido-5-azanonane (4, Ac-R-MeSPD),
a mimic of N1-acetylspermidine (an excellent substrate for
PAO), is also described. This compound and its different
enantiomers are essential to bypass the acetylation block of
R-methylated polyamine analogues by SSAT and to study the
substrate requirements of PAO. The biological tolerances of 1-3
were for the first time measured in MT-SSAT transgenic and
syngenic mice in vivo. Furthermore, these analogues were also
studied with MT-SSAT transgenic rats in vivo and with the
immortalized MT-SSAT transgenic rat fibroblasts in vitro.
Figure 1. The structures of the R-methylated polyamine analogues
used in this study: 1, R-MeSPD, R-methylspermidine; 2, R-MeSPM,
R-methylspermine; 3, R,R′-Me2SPM, bis-R,R′-methylspermine; 4, Ac-
R-MeSPD, N8-acetyl-R-methylspermidine.
which is the rate-limiting step in the interconversion of the
natural polyamines. However, 1-3 turned out to be capable of
inducing biosynthesis of SSAT similarly to spermine and
spermidine.12
Chemical Synthesis
Scheme 2 outlines the synthesis of the studied R-methylated
polyamine analogues 1-3. Synthesis was started from com-
mercially available ethyl 3-aminobutyrate (5), which was
reduced by LiAlH4 to amino alcohol 6, protected with benzyl-
chloroformate (CbzCl) to give 7, and mesylated to our key
intermediate 8. This mesyl derivative, which was not further
purified, was easily converted either to N-protected methyl-
spermidine 9, bromide derivative 10, or amino alcohol 12.
To prepare compound 1, the intermediate 8 was first aminated
with an excess of putrescine to 9 at 0-20 °C, which decreased
the number of minor byproducts that were difficult to separate
and enabled good yield (72%). The target trihydrochloride of 1
was obtained after catalytic hydrogenation and crystallization
from aqueous acidic alcohol in 46% overall yield as calculated
for starting 5.
The synthesis of corresponding spermine analogue 2 was
started from mesyl intermediate 8 by elongation of the protected
polyamine backbone first with an excess of 4-aminobutanol at
0-37 °C to give 12, which was then protected to 13, mesylated,
and treated with an excess of 1,3-diaminopropane in THF at
0-37 °C to give 14 in 70% yield. We also tried amination at
Analogues 1-3 are as effective as the natural polyamines in
inducing the conversion of the right-handed B-DNA to left-
handed Z-DNA.13 Furthermore, 1 (similarly to spermidine) and
2 (similarly to spermine due to its conversion to spermidine)
serve as substrates for deoxyhypusine synthase, which post-
translationally modifies the eukaryotic initiation factor 5A.14
Moreover, 1 and 3 proved to be metabolically quite stable, as
they are not substrates for diamine or monoamine oxidases15,16
and as such are auspicious tools to evaluate the roles of the
polyamines in different cellular functions both in vivo and in
vitro. Compound 2, on the other hand, is more complicated, as
one end of the spermine backbone is unmodified and this
analogue can serve as a substrate for the catabolic enzymes.
We have recently published results covering the effects of
these analogues in the transgenic rats.17,18 The SSAT transgene
under the control of the mouse metallothionein I promoter (MT)
is greatly induced in the MT-SSAT transgenic rats by zinc
administration. This leads to drastically reduced levels of the
higher polyamines in the pancreas and results in a severe
necrotizing pancreatitis.18 In vitro studies have shown that, in
Scheme 2. Synthesis of Racemic R-Methylated Polyamines 1-3a
a Reagents and conditions: (i) LiAlH4, THF, reflux; (ii) Cbz-Cl, H2O, NaHCO3; (iii) MsCl, Et3N, 0 °C; (iv) H2N(CH2)4NH2, THF, 0-20 °C; (v) (1)
H2/Pd, AcOH-MeOH 1:1; (2) HCl, MeOH; (vi) LiBr, THF; (vii) H2N(CH2)4OH, THF, 0-37 °C; (viii) (1) MsCl, Et3N, (2) H2N(CH2)3NH2, THF, 0-20 °C;
(ix) NsCl, Et3N, CH2Cl2, 0 °C; (x) (1) K2CO3, DMF; (2) PhSH, K2CO3, DMF.

R-Methylated Polyamine Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 401
Scheme 3. Synthesis of Enantiomers of Acetyl-R-methylspermidine, 4a
a Reagents and conditions: (i) LiAlH4, Et2O, -5 °C; (ii) Ns-Cl, Et3N, CH2Cl2, 0 °C; (iii) PhtN(CH2)4I, K2CO3, DMF; (iv) (1) HCl, EtOH, EtAc; (2)
AcCl, Et3N, CH2Cl2, 0 °C; (v) (1) PhSH, K2CO3, DMF; (2) H2NNH2‚H2O, EtOH, reflux; (3) HCl, MeOH.
20 °C, but this only increased the number of minor byproducts,
which made purification of the compound 14 more laborious.
The target tetrahydrochloride of 2 was obtained after deprotec-
tion similar to 1 in 41% overall yield calculated for N-Cbz-3-
aminobutanol 7.
The above linear strategy was ineffective for the synthesis
of R,R′-Me2SPM 3. However, due to symmetry of the target
molecule, a straightforward convergent strategy was developed
starting from bis-nosyl protected putrescine 11. This derivative
was not readily soluble in most of organic solvents, except DMF
and DMSO. The attempts to alkylate 11 with mesyl or tosyl
derivatives of 7 gave low yields. However, the bromide
derivative 10 reacted smoothly with 11 in the presence of K2-
CO3, and after selective removal of Ns-groups with a small
excess of thiophenol, symmetrical 15 was obtained in a high
yield. As previously, Cbz-groups were removed by catalytic
hydrogenation and the tetrahydrochloride of 3 was isolated in
57% overall yield as calculated for 7.
To prepare isomers (R)-4 and (S)-4, the corresponding
enatiomerically pure nitriles 16 were synthesized as described
earlier20 and were reduced by LiAlH4 without racemization to
give the key amines (R)-17 and (S)-17 (Scheme 3). These amines
were converted into nosylates 18 and then smoothly alkylated
with 4-iodophthalimidobutane to give enantiomers (R)-19 and
(S)-19 in 89% and 91% yields, respectively. Removal of the
Boc group with HCl/EtOH followed by acetylation and subse-
quent one-pot removal of nosyl and phthaloyl groups with PhSH
and H2NNH2‚H2O, respectively, resulted in (R)-4 and (S)-4,
which were converted to hydrochlorides and crystallized. In
conclusion, the earlier unknown (R)-4 and (S)-4 were prepared
in seven steps with good overall yields 51% and 52%,
respectively, as calculated for starting material 16.
Figure 2. The distribution of polyamines and their R-methylated
analogues in the liver of MT-SSAT transgenic mice and their syngenic
littermates after 24 h treatment with the indicated dosages. N ) 3-5;
1, R-methylspermidine; 2, R-methylspermine; 3, bis-R,R′-methylsper-
mine; black, spermidine; white, combined amounts of putrescine, N1-
acetylspermidine, and spermine; gray, R-methylated polyamine ana-
logues.
pronounced in the transgenic mice, but only in the transgenic
liver, 1 was detected as the degradation product of 2. On the
other hand, in the syngenic pancreas, 2 appeared to be
completely degraded as shown by the large accumulation of
spermidine. However, whether 2 degraded to 1 or spermidine
was impossible to distinguish due to the large spermidine peak
in the HPLC graphs that obscured the possible appearance of
1. Interestingly, 2 appeared to be stable in the pancreas of the
transgenic mice (Table S14 of the Supporting Information). The
spermine levels remained mostly unaffected or decreased slightly
in response to the treatments; only in the livers of the syngenic
animals and in the kidneys of both mouse lines treated with 3
were spermine levels significantly depleted. The analogues did
not affect plasma R-amylase or ALAT activities (Tables S1,
S6, and S11 of the Supporting Information). All analogues were
well-tolerated and the general tolerability was comparable to
that of the natural polyamines in mice (LD50 values (ip, as
hydrochloride) of 870 mg/kg for spermidine and 370 mg/kg
for spermine).
Biological Results and Discussion
The methylated polyamines accumulated in a dose-dependent
manner in the three tissues studied: liver (Figure 2), pancreas
(Tables S4, S9, and S14 of the Supporting Information), and
kidney (Tables S5, S10, and S15 of the Supporting Information)
of the MT-SSAT transgenic and syngenic mice. Target tissues
were selected on the basis of our focus in current research: liver
for toxicity and regeneration, pancreas due to a pancreatitis
study, and kidney because higher polyamines are associated with
nephrotoxicity. The dosages used in the studies, 50-500 mg/
kg of 1 (equaling 1-10-fold of the dosage used for the liver
regeneration or pancreatitis studies in rat), 12.5-50 mg/kg of
2, and 12.5-50 mg/kg of 3 (equaling 0.25-1-fold of the dosage
used in the pancreatitis studies in rat), were well-tolerated in
mice. The total polyamine levels were not much affected in
either mouse line by the treatments, with the exception of liver,
where spermidine was markedly depleted (Figure 2, Tables S3-
S5, S8-10, and S13-15 of the Supporting Information). In both
mouse lines the SSAT induction was greatest with 1 (2-20-
fold), whereas 3 resulted in practically no SSAT induction at
all (Tables S2, S7, and S12 of the Supporting Information). As
expected, the results from mice treated with 2 were more
complicated. The induction of SSAT by the compounds
The MT-SSAT transgenic rats were treated twice with 1-3
at 25 mg/kg dose, and none of the animals were lost during the
study. However, at higher doses the mortality rate quickly rose
(results not shown). While tissue SSAT activities were only
marginally affected, the concentrations of spermidine and
spermine were reduced, especially in the liver (Table S17 of
the Supporting Information). As in the mouse, 1 appeared to
be metabolically stable in the transgenic rat, whereas 2 was
readily degraded in both the liver and the kidney. Furthermore,
3 degraded to 1 to some extent only in the liver (Table S17 of
the Supporting Information). No degradation of spermine
analogues was detected in the rat pancreas, but again low
amounts of 1 could have remained undetected with the used
HPLC system due to the spermidine peak. As a conclusion, it
appears that there are some species-specific and/or the trans-

402 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Ja¨rVinen et al.
Figure 4. Lineweaver-Burk plot to calculate kinetic values of racemic
and different isomers of N8-acetyl-R-methylspermidine. The kinetic
studies of human recombinant polyamine oxidase were performed in
duplicates with four or five different substrate concentrations from 10
to 200 µM. Reactions were carried out in total volumes of 180 µL
containing 100 mM glycine-NaOH (pH 9.5) and 5 mM DTT and were
allowed to proceed for 10-60 min at 37 °C before HPLC analysis. No
degradation of the substrates was detected in the absence of the enzyme.
(S)-4, (S)-N8-acetyl-R-methylspermidine; 4, racemic N8-acetyl-R-me-
thylspermidine; (R)-4, (R)-N8-acetyl-R-methylspermidine; AcSPD, N1-
acetylspermidine.
Figure 3. The distribution of polyamines and their R-methylated
analogues in the immortalized rat fibroblasts during 48 h culture. Cells
were plated in triplicates in six-well culture plates in Dulbecco’s
modified Eagle’s medium supplemented with heat-inactivated 10% fetal
bovine serum and gentamycin 50 µg/mL 24 h before the growth medium
was replaced with fresh medium and the drugs. Co, untreated cells;
DENSpm, 10 µM diethylnorspermine; 1, 1 mM R-methylspermidine;
2, 10 µM R-methylspermine; 3, 1 mM bis-R,R′-methylspermine; black,
spermidine; white, combined amounts of putrescine and spermine; gray,
R-methylated polyamine analogues. No N1-acetylspermidine was
detected in either fibroblast cell lines.
µM, kcat ) 8.5 s-1), suggesting that PAO could readily use
acetylated R-isomers of R-substituted polyamine analogues.
Thus, the polyamine analogues with other alkyl and/or aromatic
substituents in the R-position are interesting compounds for
further studies to determine the substrate requirements of PAO.
In summary, the synthetic schemes described in the present
paper allow the preparation of 1-4 in gram quantities in more
than 99% purity. Suggested synthetic schemes are also suitable
for the preparation of (R)- and (S)-isomers of 1-3, which may
exhibit different biological activities. The strong stereoselectivity
of PAO, which was demonstrated for the first time with the
different isomers of 4, may have biological implications. The
presented animal experiments show that these polyamine
analogues are well tolerated in mice. Furthermore, the studies
in vitro confirm that these analogues are effectively transported
into fibroblasts and can support cell proliferation when the
natural polyamines are severely depleted. These compounds
have proven to be useful tools in studies aimed at elucidating
the functions of single polyamines both in vivo and in vitro.
genecity-related differences in the maintenance of the polyamine
homeostasis in the studied animals.
The in vitro studies with immortalized rat fibroblasts derived
from the MT-SSAT transgenic and syngenic rats showed that
the treatment with 1-3 effectively reduced the pools of natural
polyamines (Figure 3). Both 1 and 3 were well-tolerated at 1
mM, whereas concentrations higher than 10 µM of 2 were toxic.
The supplementation of the cells with 1 mM aminoguanidine
(AG) had insignificant effects on the polyamine contents of the
fibroblasts. However, 2 was readily degraded in the absence of
AG in both cell lines, while the degradation of 3 was detected
only in the transgenic cells independent of the AG treatment
(Tables S19A,B of the Supporting Information). Moreover, in
the transgenic cells, 2 clearly inhibited the proliferation of the
fibroblasts in the absence of AG, while in rodents both spermine
analogues were equally well tolerated (Table S17 of the
Supporting Information). Interestingly, the SSAT activity
increased slightly (2-10-fold) in the transgenic cell lines upon
exposure to 1-3 but remained unaffected in the control cells,
whereas DENSpm induced SSAT 200-300-fold in the trans-
genic cells but only about 10-fold in the control cells (Table
S18 of the Supporting Information). The maximal uptake of
the analogues appeared to be reachable at low concentrations
in vitro, whereas in vivo the analogue accumulation depended
on the dose (Tables S3-S5, S8-S10, S13-S15, S17, and
S19A,B of the Supporting Information).
To study the fate of 4, the use of recombinant human PAO
was essential, as R-methylated polyamine analogues are not
acetylated in vivo or in vitro. Furthermore, 4 is not effectively
transported into the immortalized fibroblasts (unpublished
observations), and in fact, acetylation is considered to facilitate
the export of excess polyamines. In addition, 4 is an excellent
compound to assess stereospecific substrate requirements for
PAO. We found that PAO strongly preferred (Figure 4) the
R-isomer of 4 (Km ) 95 µM, kcat ) 9.0 s-1) versus the S-isomer
(Km ) 170 µM, kcat ) 1.2 s-1). Expectedly, the degradation of
racemic 4 (Km ) 100 µM, kcat ) 1.3 s-1) was strongly affected
by the presence of (S)-4. Interestingly, the kcat value for (R)-4
was slightly higher than that for N1-acetylspermidine (Km ) 14
Experimental Section
Chemistry. Ethyl 3-aminobutyrate, (R)-alaninol, (S)-alaninol,
1,4-diaminobutane, 4-(N-iodobutyl)phthalimide, thiophenol, meth-
anesulfonyl chloride (MsCl), LiAlH4, and o-nitrophenylsulfonyl
chloride (NsCl) were purchased from Aldrich (United States), and
the rest of the chemicals were from Fluka (Switzerland). (R)- And
(S)-N-(tert-Butyloxycarbonyl)-3-amino-butyronitrile (16) were pre-
pared following published procedures.20 TLC was carried out on
precoated Kieselgel 60 F254 plates and column chromatography
with Kieselgel (40-63 µm, Merck, Germany) using the following
elution systems: (A) 97:3 CHCl3-MeOH, (B) 9:1 dioxane-25%
ammonia, (C) 4:2:1:2 n-butanol-AcOH-pyridine-H2O, (D) CHCl3,
(E) 200:1 CHCl3-MeOH, (F) 97:3 dioxane-25% ammonia, (G)
95:5 CHCl3-MeOH, (H) 96:4 dioxane-25% ammonia, (I) 98:2
dioxane-25% ammonia, (J) 95:5 dioxane-25% ammonia, (K) 1:2
EtOAc-hexane, and (L) 75:25 dioxane-25% ammonia. Melting
points were determined in open capillary tubes and are uncorrected.
Optical rotation angles were measured with Perkin-Elmer 241 digital
polarimeter. Chiral-HPLC analysis was performed using a Whelk-O
1 (R,R) 25 cm × 4.6 mm column, with an isocratic run from 0 to
75 min with a flow rate of 0.5 mL/min of 60% ethanol to separate
(R)- and (S)-isomers of Ac-R-MeSPD after dansylation, and
treatment was as described previously.21 1H and 13C NMR spectra

R-Methylated Polyamine Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 403
were measured on a Bruker Avance 500 DRX using tetramethyl-
silane (TMS) in CDCl3 or sodium 3-(trimethylsilyl)propanesulfonate
(TSP) in D2O as internal standards. Chemical shifts are given in
ppm, and the letter J indicates normal JHH couplings and all J
values are given in hertz.
δ 50.03 t, 48.36 d, 46.89 t, 41.85 t, 33.34 t, 26.85 t, 25.69 t, 20.38
q. Anal. (C8H24N3Cl3) C, H, N.
N-(Benzyloxycarbonyl)-3-amino-1-bromobutane (10). The
mesyl intermediate 8 was prepared as in 9 from 7 (3.01 g, 13.5
mmol), Et3N (2.52 mL, 18.1 mmol), and MsCl (1.16 mL, 15 mmol).
The evaporated and dried residue was dissolved in THF (5 mL),
and LiBr (3.5 g, 40 mmol) in THF (20 mL) was added in one
portion. The reaction mixture was stirred overnight at 20 °C,
chloroform (30 mL) was added, solids were filtered off, and the
filtrate was evaporated in vacuo. The residue was dissolved in
chloroform (50 mL); washed with water (2 × 30 mL), 1 M NaHCO3
(2 × 15 mL), and brine (20 mL); and dried (MgSO4). The
evaporated residue was dried in vacuo over P2O5/KOH to give 10
3
3-Aminobutan-1-ol (6). A solution of freshly distilled ethyl
3-aminobutyrate (5, 13.5 g, 0.103 mol) in dry THF (50 mL) was
added dropwise to the stirred suspension of LiAlH4 (8 g, 0.21 mol)
in dry THF (150 mL). The reaction mixture was refluxed with
stirring for 3 h and kept at 20 °C overnight. The residue was
quenched first with water (11.4 mL), 20% NaOH (10.6 mL), water
(29.0 mL), and finally with 40% NaOH (34.2 mL). The organic
phase was separated, and the solids were extracted with hot
chloroform (4 × 80 mL). The combined organic layers were dried
(MgSO4) and evaporated in vacuo, and the residue was distilled
(bp 108-9 °C/42 mmHg, lit.22 bp 73 °C/7 mmHg) to give 6 (7.3
g, 80%) as a colorless liquid: 1H NMR (CDCl3) δ 3.79-3.68 (2H,
m, CH2O), 3.12-3.03 (1H, m, MeCH), 2.58 (3H, bs, OH, NH2),
1.62-1.54 (1H, m, CHCH2), 1.50-1.40 [1H, m, CHCH2 (due to
chiral center on the next carbon, these CH2-protons have different
chemical shifts)], 1.09 (3H, d, J ) 6.5, CH3).
N-(Benzyloxycarbonyl)-3-aminobutan-1-ol (7) was prepared
from benzyl chloroformate (4.65 mL, 33 mmol), 6 (2.7 g, 30 mmol),
THF (30 mL), water (5 mL), and NaHCO3 (3.81 g, 45 mmol) using
the known method.23 The residue was triturated with a 1:2 ether-
hexane mixture (80 mL), and the precipitate was filtered and dried
in a vacuum over P2O5, to give 7 (6.03 g, 90%): mp 60 °C; Rf
0.38 (A); 1H NMR (CDCl3) δ 7.37-7.28 (5H, m, Ph), 5.09 (2H, s,
CH2Ph), 4.75 (1H, bs, NHCbz), 4.01-3.93 (1H, m, MeCH), 3.67-
3.59 (2H, m, CH2O), 2.98 (1H, bs, OH), 1.83-1.72 (1H, m), 1.45-
1.35 [1H, m, CHCH2 (due to chiral center on the next carbon, these
CH2-protons have different chemical shifts)], 1.20 (3H, d, J ) 6.5,
CH3).
1
(3.78 g, 95%) as a viscous oil: Rf 0.61 (D); H NMR (CDCl3) δ
7.37-7.26 (5H, m, Ph), 7.11 (1H, br s, NHCbz), 5.03 (2H, s, CH2-
Ph), 3.78-3.72 (1H, m, MeCH), 3.46-3.41 (2H, t, J ) 7.4, CH2-
Br), 2.10-1.99 [1H, m (due to chiral center on the next carbon,
these CH2-protons have different chemical shifts)], 1.95-1.85 [1H,
m, CHCH2 (due to chiral center on the next carbon, these CH2-
protons have different chemical shifts)], 1.12 (3H, d, J ) 6.6, CH3).
Bis-N1,N4-o-nitrophenylsulfonyl-1,4-diaminobutane (11). To
a cooled (0 °C) solution of freshly distilled 1,4-diaminobutane (1.1
g, 12.5 mmol) and Et3N (5.2 mL, 37.5 mmol) in dry DCM (50
mL) was added NsCl (6.05 g, 27.3 mmol) in dry DCM (30 mL)
within 40 min with vigorous stirring. After addition, stirring was
continued for 1 h at 0 °C and 3 h at room temperature. The
precipitate was filtered off, washed with MeOH (3 × 15 mL) and
chloroform (3 × 10 mL), and dried in vacuo over P2O5 to give 11
(5.3 g, 91%) as a pale-yellow crystals: Rf 0.55 (E); mp 185-6 °C;
1H NMR (DMSO-d6) δ 7.98-7.91 (4 H, m, Ph), 7.86-7.81 (4H,
m, Ph); 2.87-2.82 (4H, m, CH2N), 1.44-1.37 (4H, m, CCH2C).
N8-(Benzyloxycarbonyl)-8-amino-5-azanonan-1-ol (12). Mesyl
intermediate 8 was prepared as in 9 from 7 (3.34 g, 15 mmol),
Et3N (2.61 mL, 19 mmol), and MsCl (1.24 mL, 16 mmol). The
evaporated residue was dissolved in THF (10 mL) and cooled to 0
°C. To the resulting solution the cold (0 °C) mixture of 4-ami-
nobutanol (12.3 g, 138 mmol) and THF (20 mL) was added in one
portion, and reaction mixture was stirred at each three temperatures
(0, 20, and 37 °C) for 12 h, totaling 36 h. The solvent and an excess
of 4-aminobutanol were evaporated in vacuo, and the residue was
dissolved in 2 M NaOH (25 mL) and extracted with DCM (2 × 45
mL). The organic layer was separated, washed with brine (15 mL),
dried (Na2SO4), and evaporated in vacuo. The residue was purified
on silica gel with eluent F, which resulted in 12 (3.75 g, 85%) as
a viscous oil: Rf 0.45 (B); 1H NMR (CDCl3) δ 7.40-7.29 (5H, m,
Ph), 5.09 (2H, s, CH2Ph), 5.03 (0.5H, s, NHCbz), 5.01 (0.5H, s,
NHCbz), 3.85-3.77 (1H, m, MeCH), 3.57 (2H, t, J 6.3, CH2OH),
2.70-2.55 (4H, m, CH2N), 1.76-1.51 (8H, m, NH, OH, CCH2C),
1.18 (3H, d, J ) 6.5, CH3).
N8-(Benzyloxycarbonyl)-1,8-diamino-5-azanonane (9). To a
stirred and cooled (0 °C) solution of 7 (5.51 g, 24.7 mmol) and
Et3N (5.22 mL, 37.5 mmol) in dry DCM (60 mL) was added MsCl
(2.14 mL, 27.5 mmol) in dry DCM (10 mL) within 10 min. Stirring
was continued for 1 h at 0 °C and 30 min at room temperature,
and the reaction mixture was poured into 1 M NaHCO3 (40 mL)
solution. The organic layer was separated and washed with water
(2 × 10 mL), 0.5 M H2SO4 (3 × 35 mL), water (2 × 10 mL), and
1 M NaHCO3 (10 mL). The dried (MgSO4) and evaporated residue
of 8 was dissolved in anhydrous THF (40 mL) and cooled to 0 °C,
a cold (0 °C) solution of 1,4-diaminobutane (35.1 g, 400 mmol) in
dry THF (40 mL) was added in one portion, and the reaction
mixture was kept for 6 h at 0 °C and 16 h at room temperature.
THF and excess of 1,4-diaminobutane were evaporated in vacuo
and the residue was purified on a silica gel column using eluent B
1
to give 9 (5.3 g, 72%) as a viscous oil: Rf 0.15 (B); H NMR
(CDCl3) δ 7.37-7.27 (5H, m, Ph), 5.57 (1H, bs, NHCbz), 5.08
(2H, s, CH2Ph), 3.85-3.76 (1H, m, MeCH), 2.74-2.52 (6H, m,
CH2NH), 1.71-1.41 (6H, m, CCH2C), 1.40 (3H, bs, NH, NH2),
1.17 (3H, d, J ) 6.5, CH3); 13C NMR δ 156.04 s, 136.85 s, 128.49
s, 128.01 s (2C), 66.39 t, 49.77 t, 46.43 t, 45.96 d, 42.11 t, 36.64
t, 31.57 t, 27.43 t, 21.25 q.
Bis-N,5N8-(benzyloxycarbonyl)-8-amino-5-azanonan-1-ol (13).
Benzyl chloroformate (1.55 mL, 11 mmol) was added in three
portions with 15-min intervals to a cooled (0 °C) and vigorously
stirred mixture of 12 (2.94 g, 10 mmol), THF (20 mL), NaHCO3
(0.84 g, 10 mmol), and 2 M Na2CO3 (6 mL). Stirring was continued
for 1 h at 0 °C and 3 h at room temperature, organic layer was
separated, and the residue was extracted with chloroform (15 mL).
Organic layers were combined and evaporated to dryness in vacuo,
and the residue was dissolved in chloroform (30 mL); washed with
0.5 M H2SO4 (2 × 10 mL), water (15 mL), and 1 M NaHCO3 (2
× 10 mL); dried (MgSO4); and evaporated in vacuo. The residue
was purified on silica gel using first eluent E and then G to give
1,8-Diamino-5-azanonane Trihydrochloride (1, r-MeSPD). Pd
black in methanol (∼1 mL) was added to a solution of 9 (5.2 g,
17.7 mmol) in a mixture of AcOH-MeOH (1:1, 40 mL), and
hydrogenation was carried out at atmospheric pressure. Solids were
filtered off and washed with MeOH, and the combined filtrates
were evaporated in vacuo. The residue was dissolved in ethanol,
diluted with 7 M HCl (6.6 mL), and evaporated to dryness in vacuo,
and the residue was recrystallized from a H2O-MeOH-EtOH
mixture to give 1 (4.23 g, 89%) as colorless crystals: mp 191-2
°C (lit. mp 283 °C16 for lyophilized powder); Rf 0.45 (C); 1H NMR
(D2O) δ 3.53 (1H, m, MeCH), 3.21 (2H, m, NCH2), 3.15 (2 H, m,
NCH2), 3.07 (2H, m, NCH2), 2.16 [1H, m, CCH2C (due to chiral
center on the next carbon, these CH2-protons have different
chemical shifts)], 2.02 [1H, m, CCH2C (due to chiral center on the
next carbon, these CH2-protons have different chemical shifts)],
1.86-1.74 (4H, m, CCH2C), 1.36 (3H, d, J ) 6.5, CH3); 13C NMR
1
13 (3.94 g, 92%) a viscous oil: Rf 0.27 (A); H NMR (CDCl3) δ
7.34-7.27 (10H, m, Ph), 5.11 (2H, s, CH2Ph), 5.07 (2H, s, CH2-
Ph), 4.96 (0.5H, s, NHCbz), 4.57 (0.5H, s, NHCbz), 3.73-3.55
(3H, m, MeCH, CH2O), 3.30-3.18 (4H, m, CH2N), 1.75-1.45 (7H,
m, OH, CCH2C), 1.17-1.10 (3H, m, CH3); 13C NMR (CDCl3) δ
156.16, 155.90, 136.82, 128.52, 128.07, 128.00, 127.85, 67.07,
66.59, 62.33, 47.44, 45.41, 44.78, 44.34, 36.22, 35.13, 29.66, 25.02,
24.70, 21.32.
Bis-N,9N12-(benzyloxycarbonyl)-1,12-diamino-4,9-diazatride-
cane (14). The mesyl intermediate was prepared as in 9 from 13

404 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Ja¨rVinen et al.
(3.85 g, 9 mmol), Et3N (1.53 mL, 11 mmol), and MsCl (0.77 mL,
10 mmol). The evaporated and dried residue was dissolved in THF
(15 mL) and cooled to 0 °C, and a cold (0 °C) solution of 1,3-
diaminopropane (6.66 g, 90 mmol) in THF (5 mL) was added in
one portion. After 6 h at 0 °C and overnight at room temperature,
the reaction mixture was concentrated in vacuo. The residue was
poured into 1 M NaOH (15 mL), extracted with DCM (2 × 15
mL), and evaporated in vacuo, and the residue was purified on silica
gel using eluent H to give 14 (3.05 g, 70%) as a viscous oil: Rf
(S)-N3-(tert-Butyloxycarbonyl)-1,3-diaminobutane ((S)-17) was
prepared as (R)-17 starting from (S)-1620 to give (S)-17 as a colorless
oil (91%): [R]20D +12.0° (c 2.0, CHCl3) (lit.20 [R]20D +12.0°); 1H
NMR shifts were identical to those of (R)-17.
(R)-N1-(o-Nitrophenylsulfonyl)-N3-(tert-butyloxycarbonyl)-1,3-
diaminobutane ((R)-18). To the cooled (0 °C) solution of (R)-17
(1.43 g, 7.6 mmol) and Et3N (1.21 mL, 8.8 mmol) in dry DCM
(15 mL) was added the solution of NsCl (1.77 g, 8.0 mmol) in dry
DCM (7 mL) with stirring within 30 min. Stirring was continued
for 1 h at 0 °C, followed by washing the reaction mixture with
H2O (10 mL), 10% citric acid (15 mL), 1 M NaHCO3 (10 mL),
and brine (10 mL) and drying (MgSO4). The solvent was removed
in vacuo to afford (R)-18 (2.78 g, 98%) as a colorless solid: mp
115-6 °C (H2O-MeOH); Rf 0.21 (K); [R]20D -13.0° (c 5, CHCl3);
1H NMR (CDCl3) δ 8.14-8.10 (1H, m, Ar), 7.85-7.81 (1H, m,
Ar), 7.74-7.69 (2H, m, Ar), 6.23 (1H, bs, NsNH), 4.31 (1H, d J
) 6.5, NH), 3.80-3.69 (1H, m, MeCH), 3.33-3.22 (1H, m, CH2N),
3.01 (1H, m, CH2N), 1.78-1.69 [1H, m, CHCH2 (due to chiral
center on the next carbon, these CH2-protons have different
chemical shifts)], 1.51-1.43 [1H, m, CHCH2 (due to chiral center
on the next carbon, these CH2-protons have different chemical
shifts)], 1.37 (9H, s, CMe3), 1.11 (3H, d, J ) 6.7); 13C NMR δ
155.91, 148.07, 134.24, 133.28, 132.49, 130.70, 125.06, 79.59,
1
0.25 (F); H NMR (CDCl3) δ 7.34-7.29 (10H, m, Ph), 5.10 (2H,
s, CH2Ph), 5.07 (2H, s, CH2Ph), 4.79 (1H, s, NHCbz), 3.35-3.20
(5H, m, MeCH, CH2N), 2.73 (2H, t, J ) 6.8, CH2N), 2.66-2.53
(4H, m, CH2N), 1.70-1.35 (11H, m, NH, NH2, CCH2C), 1.16-
1.10 (3H, m, CH3); 13C NMR (CDCl3) δ 155.85, 136.88, 136.70,
128.48, 128.02, 127.92, 127.79, 67.07, 66.96, 66.48, 49.63, 47.85,
47.62, 47.17, 45.37, 44.77, 44.32, 40.54, 36.20, 35.06, 33.77, 27.25,
26.48, 25.95, 21.33.
1,12-Diamino-4,9-diazatridecanetetrahydrochloride(2,r-MeSPM)
was prepared as 1 from 14 (3.0 g, 6.2 mmol) and recrystallized
from a H2O-MeOH-EtOH mixture to give 2 (1.97 g, 88%) as
colorless crystals: mp 250-1 °C (dec) (lit.16 mp 247 °C for
1
lyophilized powder): Rf 0.13 (C); H NMR (D2O) δ 3.56-3.48
(1H, m, MeCH); 3.24-3.10 (10H, m, CH2N); 2.18-2.08 (3H, m,
NHCHCH2, CH2CH2NH2); 2.04-1.95 (1H, m, NHCHCH2); 1.85-
1.77 (4H, m, CCH2C); 1.36 (3H, d, J ) 6.6, CH3); 13C NMR (D2O)
δ 47.07, 45.41, 44.62, 44.00, 36.67, 30.47, 23.79, 22.83, 17.58,
17.34. Anal. (C11H32N4Cl4‚0.25H2O) C, H, N.
43.81, 40.81, 38.10, 28.26, 21.37. Anal. (C15H23N3O6S) C, H, N.
3
(S)-N1
-(o-Nitrophenylsulfonyl)-N -(tert-butyloxycarbonyl)-1,3-
diaminobutane ((S)-18) was prepared as (R)-18 starting from (S)-
17 to give (S)-18 as a colorless solid (99%): mp 115-6 °C (H2O-
MeOH); [R]20D +13.0° (c 5, CHCl3); 1H NMR shifts were identical
to those of (R)-18. Anal. (C15H23N3O6S) C, H, N.
Bis-N2,N13-(benzyloxycarbonyl)-2,13-diamino-5,10-diazatet-
radecane (15). Bromide 10 (3.78 g, 13.2 mmol), bis-sulfamide 11
(2.16 g, 4.7 mmol), and K2CO3 (4.3 g, 31 mmol) were stirred in
DMF (30 mL) for 48 h at room temperature, followed by addition
of a mixture of K2CO3 (3.7 g, 27 mmol) and PhSH (1.4 mL, 13.5
mmol) in DMF (10 mL). After stirring for an additional 12 h at 20
°C, salts were filtered off, the filtrate was evaporated to dryness in
vacuo, the residue was suspended in chloroform (50 mL), and solids
were filtered off and washed with chloroform (3 × 10 mL).
Combined filtrates were washed with water (2 × 30 mL), dried
(MgSO4), and evaporated to dryness in vacuo, and the residue was
purified on silica gel using eluent F, resulting in 15 (2.3 g, 70%)
as a colorless solid: Rf 0.46 (F); mp 107-8 °C; 1H NMR (CDCl3)
δ 7.36-7.26 (10H, m, Ph), 5.51 (2H, bs, NHCbz), 5.08 (4H, s,
CH2Ph), 3.85-3.74 (2H, m, MeCH), 2.73-2.65 (2H, m, CH2N),
2.64-2.54 (2H, m, CH2N), 2.54-2.45 (4H, m, CH2N), 1.72-1.40
(8H, m, CCH2C), 1.16 (6 H, d, J ) 6.5, CH3); 13C NMR (CDCl3)
δ 156.03, 136.86, 128.53, 128.05, 67.14, 66.41, 49.82, 46.45, 46.01,
36.66, 27.87, 21.28.
(R)-N1-Phthaloyl-N5-(o-nitrophenylsulfonyl)-N8-(tert-butyloxy-
carbonyl)-1,8-diamino-5-azanonane ((R)-19). The mixture of (R)-
18 (0.75 g, 2.0 mmol), N-(4-iodobutyl)phthalimide (0.725 g, 2.2
mmol), and K2CO3 (0.85 g, 6.2 mmol) in dry DMF (4 mL) was
stirred for 24 h at 20 °C. Solvent was evaporated in vacuo, the
residue was treated with EtOAc-H2O (2:1, 30 mL), and the organic
layer was separated, washed with H2O (10 mL) and brine (20 mL),
dried (MgSO4), and evaporated in vacuo. The residue was crystal-
lized from EtOAc-n-hexane to give (R)-19 as pale-yellow crystals
(1.03 g, 89%): mp 109-110 °C (EtOAc-n-hexane); [R]20D -7.0°
(c 2, EtOAc); 1H NMR (CDCl3) δ 8.00-7.96 (1H, m, Ar), 7.86-
7.80 (2H, m, Ar), 7.73-7.68 (2H, m, Ar), 7.66-7.61 (2H, m, Ar),
7.58-7.53 (1H, m, Ar), 4.48 (1H, bs, NH), 3.66 (2H, t, J ) 6.5,
CH2NPht), 3.62-3.55 (1H, m, MeCH), 3.40-3.21 (4H, m, CH2N);
1.76-1.52 (6H, m, CCH2C), 1.42 (9H, s), 1.11 (3H, d, J ) 6.5
Hz); 13C NMR δ 168.35, 133.99, 133.49, 133.33, 132.09, 131.54,
130.68, 124.10, 123.28, 78.80, 47.40, 45.14, 37.11; 36.18, 28.41,
25.70, 25.53, 21.47.
2,13-Diamino-5,10-diazatetradecane tetrahydrochloride (3,
r,r′-Me2SPM) was prepared as 2 from 15 (2.3 g, 4.6 mmol) to
give 3 (1.47 g, 85%) as colorless crystals: mp 224-5 °C (dec)
(S)-N1-Phthaloyl-N5-(o-nitrophenylsulfonyl)-N8-(tert-butyloxy-
carbonyl)-1,8-diamino-5-azanonane ((S)-19) was prepared as (R)-
19 starting from (S)-18 to give (S)-19 as a pale-yellow crystals
1
(lit.16 mp 180 °C for lyophilized powder): Rf 0.13 (C); H NMR
(91%): mp 109-110 °C (EtOAc-n-hexane); [R]20 +7.0° (c 2,
D
(D2O) δ 3.55-3.46 (2H, m, MeCH); 3.21 (4H, t, J ) 6.8, CH2N);
3.17-3.11 (4H, m, CH2N), 2.18-2.09 (2H, m, CCH2C), 2.04-
1.93 (2H, m, CCH2C), 1.84-1.75 (4H, m, CCH2C); 1.35 (6H, d, J
) 6.6, CH3); 13C NMR (D2O) δ 49.86, 48.14, 46.78, 33.27, 25.64,
20.09. Anal. (C12H34N4Cl4) C, H, N.
1
EtOAc); H NMR shifts were identical to those of (R)-19.
(R)-N1-Phthaloyl-N5-(o-nitrophenylsulfonyl)-N8-acetyl-1,8-di-
amino-5-azanonane ((R)-20). To the solution of (R)-19 (0.97 g,
1.69 mmol) in EtOAc (8 mL) was added the solution of dry HCl
in EtOH (10 M, 4 mL) and the mixture was stirred for 30 min at
20 °C. Solvents were evaporated in vacuo, the residue was
coevaporated with DMF (2 × 15 mL) in vacuo, and the residual
oil was dissolved in dry DCM (8 mL) and Et3N (1.41 mL, 10.2
mmol). After cooling (0 °C), AcCl (1.17 mL, 2.38 mmol) in dry
DCM (3 mL) was added with stirring over 10 min, and stirring
was continued for an additional 15 min at 0 °C, followed by adding
MeOH (3 mL). The solvents were evaporated in vacuo, the residue
was dissolved in EtOAc (40 mL); washed with H2O (20 mL), 10%
citric acid (10 mL), and brine (20 mL); dried (MgSO4); and
evaporated in vacuo. The residue was crystallized from (EtOAc-
n-hexane, 1:2) to give (R)-20 as a pale-yellow crystals (0.8 g, 92%):
(R)-N3-(tert-Butyloxycarbonyl)-1,3-diaminobutane ((R)-17).
To a cooled (-5 °C) suspension of LiAlH4 (0.96 g, 25 mmol) in
Et2O (20 mL) was added a solution of (R)-1620 (1.7 g, 9.2 mmol)
in Et2O (15 mL) with stirring within 20 min, and stirring was
continued for 45 min at -5 °C, followed by quenching of the
reaction mixture with 20% (w/w) aq NaOH. The organic phase
was separated, the residue was extracted with Et2O (3 × 20 mL),
and the combined organic phases were washed with brine (20 mL).
Ether was evaporated in vacuo and the residue was purified on
silica gel using eluent I to give (R)-17 (1.56 g, 90%) as a colorless
oil: Rf 0.33 (J); [R]20D -12.0° (c 2.0, CHCl3) (lit.20 [R]20D -12.0°);
1H NMR (CDCl3) δ 4.61 (1H, bs, NH), 3.77 (1H, bs, MeCH), 2.79-
2.69 (2H, m, CH2N), 1.60-1.44 (m, 2H), 1.43 (s, 9H), 1.08 (3H,
d, J ) 6.6 Hz); 13C NMR (CDCl3) δ 155.51, 78.89, 44.26, 40.99,
38.81, 28.34, 21.43.
Rf 0.54 (J); mp 125-6 °C (EtOAc-n-hexane, 1:2); [R]20 -6.7°
D
(c 2, EtOAc). 1H NMR (CDCl3) δ 7.96-7.92 (1H, m, Ar), 7.86-
7.80 (2H, m, Ar), 7.74-7.69 (2H, m, Ar), 7.66-7.61 (2H, m, Ar),
7.57-7.52 (1H, m, Ar), 5.58 (1H, d, J ) 8.1, NH); 4.00-3.89

R-Methylated Polyamine Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 405
(1H, m, MeCH); 3.67 (2H, t, J ) 6.9, CH2NPht), 3.40-3.17 (4H,
m, CH2N), 1.97 (s, 3 IÄ), 1.85-1.54 (6H, m, CCH2C), 1.14 (3H, d,
J ) 6.5 Hz); 13C NMR δ 169.94, 168.55, 148.37, 134.21, 133.60,
133.36, 132.22, 131.77, 130.63, 124.21, 123.43, 48.02, 45.47, 43.60,
37.28, 36.29, 25.85, 25.81, 23.60, 21.32.
(S)-N1-Phthaloyl-N5-(o-nitrophenylsulfonyl)-N8-acetyl-1,8-di-
amino-5-azanonane ((S)-20) was prepared as (R)-20 starting from
Recombinant Protein Studies. The human recombinant PAO
was produced according to the Qiagen Qiaexpressionist manual,
and the protein was purified under native conditions using Ni-
NTA His Bind Resin (Novagen) as described in ref 17, except
without affinity purification. PAO was estimated to be 80% pure
according to SDS-PAGE. However, the contaminating bacterial
protein did not have any PAO-like activity (data not shown). The
kinetic studies of PAO were performed in duplicates with four or
five different substrate concentrations from 10 to 200 µM. The total
volumes of reactions were 180 µL containing 100 mM glycine-
NaOH (pH 9.5), 5 mM DTT, and appropriate amount of PAO. The
reactions were allowed to proceed for 10-60 min at 37 °C before
addition of 20 µL of 100 µM diaminohexane as internal standard
in 50% w/v sulfosalicylic acid prior to HPLC analysis.
Assays Related to Biological Studies. The SSAT activity was
assayed as described earlier.24 HPLC was used to determine the
concentrations of the polyamines and their R-methylated analogues
essentially as described by Hyvo¨nen et al.25 R-Amylase and alanine
amino transferase (ALAT) were determined from heparinized
plasma of the mice using a Microlab 200 from Merck.
(S)-19 to give (S)-20 as a pale-yellow crystals (91%): mp 125-6
1
°C (EtOAc-n-hexane, 1:2); [R]20 +6.7° (c 2, EtOAc); H NMR
D
shifts were identical to those of (R)-20.
(R)-N8-Acetyl-1,8-diamino-5-azanonane ((R)-4). A mixture of
(R)-20 (0.75 g, 1.45 mmol), PhSH (0.23 mL, 2.25 mmol), and K2-
CO3 (0.62 g, 4.5 mmol) in DMF (3 mL) was stirred for 16 h at 20
°C, the solvent was removed in vacuo, and the residue was dissolved
in the mixture of DCM-H2O (3:1, 50 mL). The water layer was
separated and extracted with DCM, the combined organic phases
were washed with brine (15 mL) and dried (MgSO4), and solvent
was evaporated in vacuo. The residue was dissolved in EtOH (6
mL) containing N2H4‚H2O (0.06 g, 1.83 mmol) and refluxed for 3
h, and the solvent was evaporated in vacuo. The residue was treated
with ann EtOH-water (1:1) mixture (5 mL), an insoluble oil was
separated, and the solution was applied to Dowex 1 × 8 (200-
400 mesh, HO-form, 10 mL) and eluted with EtOH-H2O (1:1) to
get rid of phthalyl hydrazide. Fractions containing crude (R)-4 were
evaporated to dryness in vacuo, and the residue was purified on a
silica gel column using eluent L, resulting in pure (R)-4 as a base.
After treatment with HCl in EtOH and recrystallization from a
MeOH-EtOH mixture, (R)-4 (0.21 g, 72%) was obtained as
colorless crystals: Rf 0.39 (C); ee 95%; mp 200-1 °C (dec)
(MeOH-EtOH); [R]20D +4.0° (c 2, H2O); 1H NMR (D2O) δ 3.98-
3.89 (1H, m, MeCH), 3.14 (6H, m, CH2N), 2.00 (3H, s, COCH3)
1.97-1.87 [1H, m, CHCH2 (due to chiral center on the next carbon,
these CH2-protons have different chemical shifts)], 1.83-1.72 (5H,
m, 5H, CCH2C), 1.19 (3H, d, J ) 6.6, CH3); 13C NMR δ 174.00,
47.04, 44.91, 43.05, 38.96, 32.56, 24.02, 22.85, 22.05, 19.58. Anal.
(C10H25N3Cl2O) C, H, N.
Acknowledgment. We thank Sisko Juutinen, Anne Karp-
pinen, and Tuula Reponen for skillful technical assistance. This
work was supported by the Academy of Sciences of Finland
(project no. 108092) and the Russian Foundation for Basic
Research, project no. 03-04-49080.
Supporting Information Available: Biological tolerance data
and elementary analysis results of target compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
References
(1) Thomas, T.; Thomas, T. J. Polyamines in cell growth and cell
death: Molecular mechanisms and therapeutic applications. Cell. Mol.
Life. Sci. 2001, 58, 244-258.
(2) Ja¨nne, J.; Alhonen, L.; Pietila¨, M.; Keina¨nen, T. A. Genetic
approaches to the cellular functions of polyamines in mammals. Eur.
J. Biochem. 2004, 271, 877-894.
(S)-N8-Acetyl-1,8-diamino-5-azanonane ((S)-4) was prepared
as (R)-4 starting from (S)-20 to give (S)-4 (70%): ee 95%; mp
200-1 °C (dec) (MeOH-EtOH); [R]20D -4.0° (c 2, H2O); 1H NMR
shifts were identical to those of (R)-4. Anal. (C10H25N3Cl2O) C, H,
N.
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Transgenic Animal Studies. The production of the transgenic
mice and rats has been described earlier in detail.17 Both animal
lines were produced by the standard pronuclear injection technique
using the same transgene construct. The methylated polyamine
analogues were administered in saline. Three to four month old
male mice were injected once with 0, 50, 100, 150, 250, 375, or
500 mg/kg (1) or with 0, 12.5, 25, 37.5, or 50 mg/kg (2 and 3)
intraperitoneally 24 h before sacrifice. Ten week old transgenic
male rats were treated twice with 25 mg/kg (1-3) intraperitoneally
22 and 16 h before sacrifice. Tissue samples were frozen in liquid
nitrogen and homogenized in the standard buffer (25 mM Tris-
HCl pH 7.4, 0.1 mM EDTA, 1 mM DTT). An aliquot of the
homogenates was used for the polyamine assays. The homogenates
were centrifuged (at 13 000g, for 30 min, at 4 °C) and the
supernatant fractions were used for the enzyme activity assays. The
Institutional Animal Care and Use Committee of the University of
Kuopio and the Provincial Government approved the animal
experiments.
Immortalized Rat Fibroblast Studies. The production of the
immortalized fibroblasts was explained earlier in detail.17 The cells
were plated in triplicates in six-well culture plates in Dulbecco’s
modified Eagle’s medium (Invitrogen) supplemented with heat-
inactivated 10% fetal bovine serum and gentamycin (50 µg/mL;
Gibco). The cells were allowed to adhere for 24 h before the growth
medium was replaced with fresh medium and the drugs. After the
incubation, the cells were washed with PBS, detached with trypsin,
and counted, and the polyamines were determined after sulfosali-
cylic acid precipitation from the supernatant fractions with the aid
of the HPLC.
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