The use of novel C-methylated spermidine derivatives to investigate the regulation of polyamine metabolism

Article abstract of DOI:10.1021/jm200293r

The polyamines are organic polycations present at millimolar concentrations in eukaryotic cells where they participate in the regulation of vital cellular functions including proliferation and differentiation. Biological evaluation of rationally designed

Full text of DOI:10.1021/jm200293r

ARTICLE
pubs.acs.org/jmc
The Use of Novel C-Methylated Spermidine Derivatives To Investigate
the Regulation of Polyamine Metabolism
Mervi T. Hyv€onen,*,†,§ Tuomo A. Kein€anen,^,§ Maxim Khomutov,‡ Alina Simonian,‡ Janne Weisell,^
Sergey N. Kochetkov,‡ Jouko Veps€al€ainen,^ Leena Alhonen,† and Alex R. Khomutov‡
†A.I. Virtanen Institute for Molecular Sciences, Biocenter Kuopio, University of Eastern Finland, Kuopio Campus, Yliopistonranta 1E,
70210 Kuopio, Finland
‡Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow 119991, Russia
^Department of Biosciences, Biocenter Kuopio, University of Eastern Finland, Kuopio campus, Yliopistonranta 1E, 70210 Kuopio,
Finland
S Supporting Information
b
ABSTRACT: The polyamines are organic polycations present
at millimolar concentrations in eukaryotic cells where they
participate in the regulation of vital cellular functions including
proliferation and differentiation. Biological evaluation of ration-
ally designed polyamine analogs is one of the cornerstones of
polyamine research. Here we have synthesized and character-
ized novel C-methylated spermidine analogs, that is, 2-methyl-
spermidine, 3-methylspermidine, and 8-methylspermidine.
3-Methylspermidine was found to be metabolically stable in
DU145 cells, while 8-methylspermidine was a substrate for
spermidine/spermine N1-acetyltransferase (SSAT) and 2-methylspermidine was a substrate for both SSAT and acetylpolyamine
oxidase. All the analogs induced the splicing of the productive mRNA splice variant of SSAT, overcame growth arrest induced by
72-h treatment with ornithine decarboxylase (ODC) inhibitor R-difluoromethylornithine, and were transported via the poly-
amine transporter. Surprisingly, 2-methylspermidine was a weak downregulator of ODC activity in DU145 cells. Our data
demonstrates that it is possible to radically alter the biochemical properties of a polyamine analog by changing the position of the
methyl group.
’ INTRODUCTION
cytotoxicity. Some of the terminally bis-N-alkylated derivatives of
3a are at different phases of clinical trials.4 There is also a growing
interest to develop metabolically stable polyamineꢀdrug con-
jugates, which would deliver the cytotoxic drug into tumor tissue
via polyamine transporter.6 Therefore, the design of novel
chemical compounds controlling the metabolism of the enzymes
and proteins involved in the regulation of polyamine homeostasis
is of importance and may even contribute to practical polyamine-
based therapy.
Investigation of the cellular functions of 2a and 3a at the
molecular level is rather complicated due to their feasible
interconversion and their ability to substitute for each other in
some functions.1ꢀ3 Considerable original data have been pro-
vided with the aid of cells and microorganisms that are deficient
in the key enzymes of polyamine metabolism. One of the recent
examples is the construction of E. coli strains being deficient in all
the enzymes of polyamine biosynthesis.7 The use of specific
enzyme inhibitors is an efficient approach in vitro, but the
necessity to inhibit several enzymatic transformations leads to
The importance of the polyamines putrescine (Put, 1),
spermidine (Spd, 2a), and spermine (Spm, 3a) (Figure 1) for
diverse physiological functions in living organisms has been
elucidated during the past decades, but the distinct roles of 2a
and 3a are still not completely understood.1ꢀ4 Multilevel regula-
tion of not only activities but also the biosynthesis and degrada-
tion of the enzymes of polyamine metabolism, as well as
regulation of polyamine uptake and excretion, underline the
importance to develop methods either to specifically silence or
induce the required regulatory pathway. Mutants and genetically
modified microorganisms and cell lines,1ꢀ3 as well as transgenic
animals,5 are widely used for these purposes.4 However, chemical
compounds specifically “switching off” or inducing regulatory
pathways of polyamine metabolism are limited, and the most
well-known are terminally bis-N-alkylated derivatives of 3a.
Changing the structure of the N-alkyl substituent facilitated the
transition from actively transported inducers to superinducers of
spermidine/spermine N1-acetyltransferase (SSAT) that together
with efficient downregulation of ornithine decarboxylase (ODC)
and S-adenosyl-L-methionine decarboxylase (AdoMetDC) re-
sulted in polyamine depletion-induced growth arrest or
Received: March 13, 2011
Published: May 18, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Syntheses of Novel Spermidine Derivatives 5a and
6a
Scheme 2. Synthesis of Novel Spermidine Derivative 7a
Figure 1. Structures of the compounds.
(i) NaCN/DMSO; (ii) LiAlH4/Et2O/ꢀ5 ꢀC; (iii) NsCl/DCM/Et3N;
(iv) (1) CbzNH(CH2)3Br/DMF/K2CO3/45 ꢀC; (2) PhSH/DMF/
K2CO3; (v) (1) HCl/EtOH; (2) NaOH/H2O/DCM; (3) H2/Pd/
AcOH/MeOH; (4) HCl/EtOH.
cumbersome combinations of the inhibitors.8,9 A relatively simple
method to discriminate the cellular effects of 2a and 3a is the
depletion of the polyamine pool and subsequent application of
functionally active metabolically stable mimetics of 2a and 3a.
Earlier studies implied that polyamine metabolism could be
selectively modulated by the use of 1-methylated polyamine
analogs, which can fulfill many cellular functions of polyamines.
1-Methylspermidine (1-MeSpd, 4a) and 1,12-bis-methylsper-
mine (1,12-Me2Spm, 3b) restored early liver regeneration
after partial hepatectomy, prevented acute pancreatitis caused
by depletion of polyamine pool in SSAT-transgenic rats,10
and overcame growth inhibition caused by 3-d treatment with
R-difluoromethylornithine (DFMO).11 The use of optical iso-
mers of 4a revealed that only the (S)-isomer was a substrate for
deoxyhypusine synthase and capable of supporting the growth of
DU145 cells during prolonged polyamine deprivation12 or
growth of Saccharomyces cerevisiae polyamine auxotrophs.13 In
addition, the distinct optical isomers of 4a and 3b were found to
differ in their ability to regulate the biosynthesis of SSAT,
AdoMetDC, and antizyme (OAZ), a protein regulating ODC
half-life and polyamine transport.14
Here, we describe novel possibilities for the regulation of
polyamine metabolism using C-methylated derivatives of 2a
(Figure 1). Among these analogs, 2-methylspermidine (2-Me-
Spd, 5a) displayed catabolic instability toward SSAT and
acetylpolyamine oxidase (APAO), whereas 3-methylspermidine
(3-MeSpd, 6a) was metabolically stable. Our results demonstrate
that the biochemical properties of these analogs are determined
by the triprotonated backbone and by the position of the methyl
substituent.
Table 1. The Analogs as Substrates for Mouse Recombinant
SSAT
substrate
Km (μM)
Vmax (μmol/(min mg))
3
2a
4a
5a
6aa
7a
151 ( 15
4.28 ( 0.13
b
b
b
132 ( 7
78 ( 3
0.77 ( 0.01
b
7.35 ( 0.10
a 6a Ki = 52 ( 18 μM. b Not acetylated.
resulted in target trihydrochlorides 5a and 6a in two steps with
overall yields of 16% and 32%, respectively (Scheme 1).
The synthesis of 7a was more complicated and was performed
in seven steps (Scheme 2), starting from 3-[N-(tert-butyloxycar-
bonyl)]amino-1-butyl methanesulfonate (10), which was first
converted into nitrile 11, while subsequent LiAlH4 reduction
gave amine 12. Thus obtained 12 was nosylated, and the resulting
sulfonamide 13 was alkylated with 3-(N-benzyloxycarbo-
nyl)amino-1-bromopropane that after removal of the nosyl
group gave the intermediate Boc-Cbz-triamine 14. Stepwise
removal of Boc and Cbz groups resulted in target 7a with an
overall yield of 31%, as calculated from the starting methanesul-
fonate 10.
Catabolic Stability of the Analogs. Nearly 30 years ago, a
family of gem-dimethylated derivatives was synthesized, and
among 1,1-bis-methylspermidine (1,1-Me2Spd, 4b), 2,2-bis-
methylspermidine (2,2-Me2Spd, 5b), 3,3-bis-methylspermidine
(3,3-Me2Spd, 6b), 8,8-bis-methylspermidine (8,8-Me2Spd, 7b),
and 5,5-bis-methylspermidine (5,5-Me2Spd) (Figure 1), only 7b
was a substrate for SSAT.16 Here we demonstrate that the
reduction of the number of substituents dramatically affects the
substrate properties of the analogs for SSAT. Like the earlier
studied 7b, 8-methylspermidine (8-MeSpd, 7a) was a substrate
for mouse recombinant SSAT with kinetic parameters reflecting
increased affinity and catalytic activity than those observed with
2a (Table 1). Surprisingly, 5a, in contrast to 5b, was a substrate of
SSAT, whereas 6a, like the earlier 4a,11 exhibited no substrate
properties but was a competitive inhibitor of the enzyme. When
’ RESULTS AND DISCUSSION
Chemistry. There are various synthetic strategies to build up
CꢀN bonds to polyamines based upon a comprehensive review
of this area.15 In the present paper from the variety of methods,
we selected two, the Michael addition of amine to unsaturated
nitriles to prepare 5a and 6a and alkylation of the o-nitrophenyl
sulfonates with alkyl halides to prepare 7a. Hence, the addition of
an excess of putrescine to either methacrylonitrile (8a) or
crotononitrile (8b) with subsequent reduction of the intermedi-
ate diaminonitriles 9a and 9b with hydrogen over Raney-Ni
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Journal of Medicinal Chemistry
ARTICLE
Figure 2. The analogs as substrates for human recombinant APAO with
or without (w/o) 5 mM benzaldehyde (BA). Values were determined
using 0.1 or 1 μg of enzyme and 0.5 or 1 mM substrate and incubation
time of 5ꢀ60 min. Data are means ( SD, n = 6.
Figure 4. Uptake competition with [14C]-labeled 2a. DU145 cells were
incubated in serum-free medium for 10 min in the presence of 10 μM
[14C]-2a and 1, 10, or 100 μM analogs as competitors. Data are means (
SD, n = 3.
the substrate specificity of this enzyme, while in the presence of
benzaldehyde, 5a was even a better substrate of APAO than 2a
(Figure 2). Thus, our data reveal two structurally related classes
of analogs, one class demonstrating increased catabolic instability
(5a) and the other considerable catabolic stability (6a) toward
polyamine-metabolizing enzymes.
Serum amine oxidases degrade extracellular polyamines and
many of their analogs to toxic metabolites. The study of the
stability of C-methylated analogs of 2a in cell culture medium
showed that at 10 μM none of the analogs was toxic to DU145
cells (data not shown) and at 100 μM concentration only 2a and
7a produced toxic metabolites (Figure 3). Since we preferred to
use higher concentrations of the analogs in order to efficiently
replace the natural polyamines, cells were cultured in the
presence of 1 mM aminoguanidine (AG), an inhibitor of serum
amine oxidases.
Figure 3. Cytotoxicity of the degradation products of the analogs
produced by the action of bovine serum amine oxidases. MTT assay
of DU145 cells grown for 48 h with 100 μM analogs, with (black bars) or
without (white bars) 1 mM AG. Results are means ( SD, n = 8; ///
indicates statistical significance of p < 0.001 compared with “w AG”.
The Analogs Compete with 2a for Uptake. The polyamine
transporter allows the uptake of not only the natural polyamines
but also many analogs resembling the natural polyamines in
structure.20 We found here that all tested C-methylated analogs
of 2a competed for the uptake with [14C]-2a by DU145 cells
during 10-min incubation with varying efficiencies (Figure 4).
Only 5a was as efficient as unlabeled 2a in reducing the
accumulation of [14C]-label, and 6a was clearly the weakest
competitor. Thus, the polyamine transport system recognized all
tested analogs as polyamines but with different affinities or
velocities. However, the C-methylation affected uptake by the
polyamine transporter only moderately, compared with the
numerous other N-alkylated analogs, such as the N1-anthrace-
nenylmethyl homospermidine derivatives, where the position
and the size of the N-substituent had dramatic effects on the
polyamine transporter targeting.6
Compound 6a Is Not Metabolized to 3-MeSpm. The gem-
dimethylated derivatives of 2a (Figure 1) were not substrates of
spermine synthase (SpmSy) even at 5 mM concentrations.16
However, the decrease of the number of the substituents resulted
in the biosynthesis of the corresponding C-methylated Spm
analogs from 4a, 5a, and 7a in DU145 cells (Table 2). 1-MeSpm
(3c) and 2-MeSpm accumulated in cells after 24 h incubation in
the presence of the corresponding 2a analog, while 5-MeSpm
(3d) (according to IUPAC nomenclature this is 3d, not
8-MeSpm) was detectable only after 48 h (Table 2). The
biosynthesis of 3d was somewhat unexpected, because in chick
embryos the formation of only 7a from 1-methylputrescine was
detected.21 The absence of any 3-MeSpm even after 72 h
incubation of DU145 cells with 6a was very surprising. Our data
SSAT was induced in DU145 cells by incubating the analog-
preloaded cells with N1,N11-diethylnorspermine (DENSpm),
acetylated derivatives of 5a and 7a were found intracellularly
(Table S1, Supporting Information). In addition, all analogs were
excreted into the media and 7a was also in an acetylated form,
even without DENSpm treatment.
A recent X-ray study of the SSAT complex with 3a showed that
a methyl group at the third position may affect hydrophobic
contacts of the analog with the side chains of Leu128 and
Trp154, which are positioned close to the secondary (N-4)
amino group of substrate.17 In addition, this methyl group may
influence the structure of a “proton wire”, which is formed by
water molecules coordinated by the substrate amino groups and
the side chains of Asp93 and Glu92, which cover not only the
acetylated amino group, but also the neighboring N-4 amino
group. These properties apparently restrict the N1-acetylation of
6a, making it a novel catabolically stable derivative of 2a.
Earlier studies showed that a chemically synthesized mono-
acetylated derivative of 4a was a good substrate of APAO.18 In
the presence of aromatic aldehydes, APAO degraded not only 2a
and 3a19 but also 4a.18 Here, we show that benzaldehyde
efficiently stimulated APAO-dependent degradation of all the
analogs tested, with the exception of 6a (Figure 2). Methyl group
at the third position of 2a may either restrict the splitting of the
proton from third carbon atom, which is required to form the
intermediate Schiff base, or interfere with the proper binding of
the analog, which may explain the stability of 6a toward APAO in
the presence of benzaldehyde. Surprisingly, 5a was degraded by
APAO even in the absence of benzaldehyde, which is atypical for
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Table 2. Effect of the Analogs on Polyamine Content in DU145 Cells after 24, 48, and 72 h of Culturea
intracellular polyamine (pmol/μg DNA)
treatment
1
2a
3a
2b
analog
MeSpme
analogþ MeSpm
24 h
48 h
72 h
b
b
b
b
control
4a
11 ( 2
154 ( 7
15 ( 0c
39 ( 0c
23 ( 1c
30 ( 2c
122 ( 11
62 ( 3c
67 ( 5c
92 ( 4c
83 ( 3c
b c
,
264 ( 12
133 ( 4
181 ( 5
257 ( 5
37 ( 2
301 ( 14
200 ( 17
181 ( 5
257 ( 5
5a
<5c
<5c
67 ( 13
b
6a
b c
b
7a
,
15 ( 2d
b
b
b
b
control
4a
7 ( 1
n.d c
<5c
137 ( 5
11 ( 1c
38 ( 5c
18 ( 1c
18 ( 1c
133 ( 4
55 ( 1c
72 ( 12c
103 ( 2c
57 ( 4c
239 ( 6
135 ( 12
168 ( 6
287 ( 16
44 ( 2
283 ( 8
202 ( 19
168 ( 6
326 ( 22
5a
77 ( 7
b c
b
6a
,
b c
7a
,
7 ( 1d
39 ( 6
b
b
b
b
control
4a
6 ( 0
137 ( 3
16 ( 4c
38 ( 1c
22 ( 1c
20 ( 1c
154 ( 7
74 ( 24c
75 ( 4c
115 ( 2c
70 ( 2c
b c
,
311 ( 76
134 ( 6
176 ( 3
332 ( 14
60 ( 8
371 ( 84
214 ( 13
176 ( 3
5a
<5c
80 ( 7
b c
b
6a
,
b c
7a
,
4 ( 1d
43 ( 3
375 ( 17
a Cells were cultured in the presence of 100 μM analogs. All plates contained also 1 mM AG. Results are means ( SD, n = 3. b Not detectable. c Statistical
significance of p < 0.001 compared with AG-treated control group. d Ac-8-MeSpd. e MeSpm was quantified using 3c standards.
emphasize the necessity of high complementarity between the
central part of the 2a molecule and the corresponding region of
SpmSy responsible for binding of the substrate(s) and their
analogs. The lack of such a complementarity renders 6a meta-
bolically stable.
The Analogs Differ in Their Ability To Downregulate ODC.
Incubation of DU145 cells with any of the tested analogs for 24,
48, or 72 h had negligible effects on the activities of AdoMetDC,
APAO, and SSAT. Some reduction in SMO activity was observed
with 6a (Table 3). However, the most pronounced effects were
observed in the case of ODC, the activity of which was down-
regulated by all analogs tested, except 5a. Since OAZ protein is
the key regulator of ODC half-life and polyamines are known to
stimulate OAZ biosynthesis via promotion of ribosomal
frameshifting,22,23 our data suggests that 5a may be incapable
of inducing OAZ.
all tested analogs stimulated the productive splicing of SSAT
mRNA (Figure 5), and the efficiency of the analogs correlated
with the total intracellular higher polyamine concentration
(2aþ3aþanalog; R = 0.90, p < 0.01). Thus, the application of
5a provides an opportunity to induce the productive splicing of
SSAT pre-mRNA without a significant effect on ODC.
The Analogs Support Growth of DFMO-Treated DU145
Cells. Earlier studies indicated that gem-dimethylated Spd analogs
were able to overcome growth inhibition caused by a 3-d DFMO
treatment of SV-3T3 cells.16 As expected, all tested analogs
supported the growth of DFMO-treated DU145 cells and exhib-
ited about the same efficacy as 2a (Table 4). Thus, among the
analogs, 6a is not only metabolically stable but also a functional
mimetic of 2a, which makes thiscompound an exceptionally useful
instrument to investigate the cellular functions of 2a.
We also measured the intracellular levels of S-adenosyl-L-
methionine (SAM) and decarboxylated S-adenosyl-L-methionine
(dc-SAM) after 3-d culture with AG and DFMO (Table 4).
Increased dc-SAM levels in DFMO-treated cells were reduced
by treatment with the analogs, and 5a was as effective as natural
spermidine in reducing dc-SAM level.
The Analogs Modulate Alternative Splicing of SSAT Pre-
mRNA. The alternative splicing of SSAT mRNA is regulated by
intracellular polyamine level24 but the molecular mechanisms of
this regulatory pathway, as well as the mechanisms of frameshift-
ing of OAZ mRNA, still remain unclear. The alternative splicing
of SSAT pre-mRNA yields one variant encoding catalytically
active protein (SSAT) and the other containing an additional
exon (SSAT-X), targeting the mRNA to rapid degradation via a
nonsense-mediated mRNA decay pathway. Our previous studies
have shown that many but not all polyamine analogs are able to
induce the productive splicing of SSAT.24,25 Here we found that
’ CONCLUSIONS
In conclusion, our data demonstrates that functional recogni-
tion of 2a analogs isbasednot only on electrostaticinteractions but
also on proper spatial organization of the essential recognition
part(s) of the 2a molecule. Therefore, by changing the position of
the methyl group in the 2a backbone, it is possible to design novel
compounds that are suitable to study the cellular functions of the
individual polyamines. These results also provide considerable
insight into the structural limitations that must be met for the
design of effective drugs based on the polyamine backbone.
’ EXPERIMENTAL SECTION
Materials. DFMO was from ILEX Oncology Inc. (U.S.A.), [14C]-
labeled compounds from GE Healthcare, and other reagents from
Sigma-Aldrich. The synthesis of 4a is described previously.18
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ARTICLE
Table 3. The Activity of the Enzymes of Polyamine Meta-
bolism after 24, 48, or 72 h Incubation of DU145 Cells with
the Analogsa
Table 4. Effect of the Analogs on Cell Growth and SAM and
dc-SAM Levels in DU145 Cells after 72 h of Culture
treatment growth (%) SAM (pmol/106 cells) dc-SAM (pmol/106 cells)
100 ( 4b
49 ( 2
84 ( 3
80 ( 5
<5b
263 ( 14
<5b
49 ( 8b
<5b
109 ( 4b
60 ( 3b
activity (pmol/h per μg DNA)
control
DFMO
time and treatment
ODC
AdoMetDC SSAT
24 h
SMO
APAO
DFMOþ2a 89 ( 4b
DFMOþ4a 84 ( 6b
DFMOþ5a 86 ( 2b
DFMOþ6a 91 ( 5b
DFMOþ7a 93 ( 3b
126 ( 5c
177 ( 25b
148 ( 3b
149 ( 7b
138 ( 4b
control
4a
29.3 ( 1.4
61 ( 5
59 ( 1 493 ( 22 540 ( 61
67 ( 3c 362 ( 41c 545 ( 7
56 ( 3 394 ( 19 562 ( 17
62 ( 2 204 ( 20b 528 ( 57
65 ( 3 515 ( 78 562 ( 97
4.3 ( 0.3b 64 ( 2
21.7 ( 1.4b 67 ( 1
8.8 ( 0.3b 53 ( 2
4.7 ( 0.5b 53 ( 4
5a
a Cells were cultured in the presence of 100 μM analogs and 5 mM
DFMO. All plates contained also 1 mM AG. Results are means ( SD,
n = 3. b Statistical significance of p < 0.001 compared with AGþDFMO-
treated group. c Statistical significance of p < 0.01 compared with
AGþDFMO-treated group.
6a
7a
48 h
control
4a
17.7 ( 2.6
4.4 ( 0.3b 72 ( 3
16.6 ( 0.7 71 ( 2
73 ( 1
58 ( 0 680 ( 29 699 ( 21
61 ( 3 695 ( 44 779 ( 32
59 ( 3 655 ( 39 719 ( 44
5a
6a
9.3 ( 0.8b 60 ( 0b 59 ( 2 474 ( 29d 693 ( 6
7a
5.5 ( 0.6b 67 ( 2
62 ( 6 738 ( 89 770 ( 60
72 h
control
4a
7.5 ( 0.9
72 ( 6
62 ( 4 742 ( 10 662 ( 48
66 ( 5 754 ( 27 745 ( 43
61 ( 6 608 ( 42 732 ( 79
3.5 ( 0.1b 75 ( 5
9.1 ( 0.3c 79 ( 3
5a
6a
5.4 ( 0.4d 58 ( 1d 69 ( 4 571 ( 62d 775 ( 73
3.8 ( 0.3b 63 ( 1
7a
61 ( 3 832 ( 45 737 ( 99
a Cells were cultured in the presence of 100 μM analogs. All plates
contained also 1 mM AG. Results are means ( SD, n = 3. b Statistical
significance of p < 0.001 compared with AG-treated control group at
each time point. c Statistical significance of p < 0.05 compared with AG-
treated control group at each time point. d statistical significance of
p < 0.01 compared with AG-treated control group at each time point.
Figure 5. Effect of the analogs on the alternative splicing of SSAT.
Mouse primary fetal fibroblasts were treated with CHX (10 μg/mL) for
1 h and then with 100 μM analogs and CHX for 7 h. The ratio of SSAT-
X/total SSAT mRNA was measured with quantitative RT-PCR. Data are
means ( SD, n = 3. C, control; / and /// indicate statistical significance
of p < 0.05 and 0.001, respectively, compared with CHX-treated group.
Syntheses. 1,4-Diaminobutane, thiophenol, LiAlH4, NaCN, o-nit-
rophenylsulfonylchloride (NsCl), and anhydrous LiBr were purchased
from Aldrich (U.S.A.), and the rest of chemicals were purchased from
Fluka (Switzerland). 3-[N-(tert-Butyloxycarbonyl)]amino-1-butyl
methanesulfonate and 3-(N-benzyloxycarbonyl)amino-1-propyl metha-
nesulfonate were prepared as recently published.26 Syntheses of 3-(N-
benzyloxycarbonyl)amino-1-bromopropane is described in the Support-
ing Information. TLC was carried out on precoated Kieselgel 60 F254
plates, and column chromatography was performed with Kieselgel
(40ꢀ63 μm, Merck, Germany) using the following elution systems:
(A) 8:2 dioxaneꢀ25% NH4OH, (B) 7:3 dioxaneꢀ25% NH4OH, (C)
4:2:1:2 n-BuOHꢀAcOHꢀPyꢀH2O. Flash chromatography was per-
formed on Kieselgel (40ꢀ63 μm, Merck, Germany), and systems for
elution are indicated in the text. Melting points were determined in open
EtOH (50 mL) was hydrogenated at atmospheric pressure for 24 h. The
catalyst was filtered off, the residue was washed with abs. EtOH, and the
combined filtrates were evaporated to dryness in vacuo to yield crude 5a,
which was purified by column chromatography on a silica gel (130 g)
eluting subsequently with solvent mixtures A and B. The crude triamine 5a
was repurified on a silica gel (70 g) eluting as above with solvent mixtures A
and B. The free base of 5a was dissolved in the mixture of EtOH (15 mL)
and 5 M HCl (5 mL) and evaporated to dryness in vacuo, which after
coevaporation with abs. EtOH and crystallization from abs. EtOH gave 5a
(0.69 g, 34%); mp 189ꢀ192 ꢀC; Rf 0.22 (C); Rf 0.16 (B). 1H NMR(D2O):
δ 3.19ꢀ2.90 (8H, m, 4*NCH2); 2.39ꢀ2.28 (1H, m, CH); 1.85ꢀ1.72 (4H,
m, CCH2CH2C); 1.16 (3H, d, J = 6.8 Hz, CH3). 13C NMR (D2O):
δ 53.48, 50.59, 45.23, 41.67, 32.27, 26.79, 25.47, 17.07. Anal. C, H, N.
3-Methyl-8-amino-4-azaoctanenitrile (9b). Prepared as 9a starting
from 1,4-diaminobutane (58.9 g, 0.67 mol) and 8b (8.97 g, 0.134 mol) to
give after distillation 9b (17.6 g, 84.7%); bp 102ꢀ103 ꢀC/0.75 mmHg;
nD20 1.4688. TLC, NMR, and anal. C, H, N were conducted.
1,8-Diamino-3-methyl-4-azaoctane trihydrochloride (6a), 3-Me-
Spd. Following the procedure for 5a and starting from 9b (1.7 g,
10.97 mmol) and a suspension of Raney nickel (10 mL) in abs. EtOH
(50 mL) provided the target 6a (1.11 g, 37.8%); mp 231ꢀ232 ꢀC; Rf
0.24 (C); Rf 0.10 (B). 1H NMR (CDCl3): δ 3.48ꢀ3.41 (1H, m, CH),
3.23ꢀ3.00 (6H, m, 3*NCH2), 2.25ꢀ2.15 (1H, m, CHCH2), 2.01ꢀ1.91
(1H, m, CHCH2), 1.85ꢀ1.75 (4H, m, CCH2CH2C), 1.36 (3H, d, J =
6.7 Hz, CH3). 13C NMR (CDCl3): δ 54.88, 47.10, 41.68, 38.75, 33.07,
26.84, 25.83, 17.96. Anal. C, H, N.
1
capillary tubes and are uncorrected. H and 13C NMR spectra were
measured on a Bruker Avance 500 DRX (Germany) using tetramethyl-
silane (TMS) in CDCl3 or sodium 3-(trimethylsilyl)-1-propanesulfo-
nate (TSP) in D2O as internal standards. Chemical shifts are given in
ppm, the letter “J” indicates normal 3JHH couplings, and all J values are
given in hertz. All final compounds were g99% purity confirmed with
analytical HPLC used according to the published method.27
2-Methyl-8-amino-4-azaoctanenitrile (9a). A solution of 1,4-diami-
nobutane (55.4 g, 0.63 mol) and 8a (17.16 g, 0.256 mol) in EtOH (8 mL)
was stirred for 2 h at 20 ꢀC, 2 h at 60 ꢀC, and 2.5 h at 90 ꢀC. Subsequent
distillation of the reaction mixture gave 9a (18.8 g, 47%). Bp 101 ꢀC/0.5
mmHg; nD20 1.4646. TLC, NMR, and anal. C, H, N were conducted.
1,8-Diamino-2-methyl-4-azaoctane trihydrochloride (5a), 2-MeSpd.
A suspension of Raney nickel (10 mL) and 9a (1.5 g, 9.7 mmol) in abs.
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4-[N-(tert-Butyloxycarbonyl)]-aminovaleronitrile (11). Finely pul-
verized NaCN (4.5 g, 92 mmol) was added in small portions to a stirred
solution of 3-[N-(tert-butyloxycarbonyl)]amino-1-butyl methanesulfo-
nate (10) (4.8 g, 18 mmol) in abs. DMSO (20 mL) and stirring was
continued for 8 h at 45 ꢀC. The reaction mixture was cooled to 0 ꢀC, then
2 M NaOH (75 mL) was added, and the resulting mixture was extracted
with Et2O (4 ꢁ 20 mL). Combined Et2O extracts were washed with
brine (15 mL), dried (MgSO4), filtered, and concentrated in vacuo. The
residue was dried at 0.5 mmHg at 30 ꢀC to afford 11 as an oil (4.73 g,
99%), which self-solidified upon the addition of a few crystals of 11.
Treatment with hexane gave 11 (3.39 g, 95.2%) as a low melting solid
(45 ꢀC). TLC, NMR, and anal. C, H, N were conducted.
4-[N-(tert-Butyloxycarbonyl)]-1,4-diaminopentane (12). A solution
of 11 (4.46 g, 22.5 mmol) in Et2O (35 mL) was added with mechanic
stirring for 20 min to a cooled (ꢀ10 ꢀC) suspension of LiAlH4 (2.33 g,
61 mmol) in Et2O (50 mL) and stirring was continued for 1 h at ca.
ꢀ5 ꢀC. The reaction mixture was cooled to ꢀ10 ꢀC and carefully
quenched subsequently with H2O (3.3 mL), 20% aq NaOH (3.1 mL),
H2O (8.5 mL), and 40% aq NaOH (10.0 mL), maintaining the
temperature below ꢀ5 ꢀC. On warming to 20 ꢀC, the organic layer
was separated, and the residue was treated with Et2O (3 ꢁ 30 mL). The
combined Et2O extracts were washed with 1 M NaHCO3 (15 mL), H2O
(5 mL), and brine (15 mL) and dried (K2CO3). Solvent was distilled off
in vacuo, and the residue was dried in vacuo over P2O5/KOH to give 12
(3.33 g, 75%) as a colorless oil. TLC, NMR, and anal. C, H, N were
conducted.
1-(N-o-Nitrophenylsulfonyl)amino-4-[N-(tert-butyloxycarbonyl)]-
aminopentane (13). A solution of NsCl (1.6 g, 7.3 mmol) in DCM
(10 mL) was added over 30 min with stirring to a cooled (0 ꢀC) solution
of 12 (1.54 g, 7.6 mmol) and Et3N (1.23 mL, 8.8 mmol) in DCM
(20 mL), and stirring was continued for 1 h at 0 ꢀC and 3 h at 20 ꢀC. The
precipitate was filtered off, the filtrate was washed with 1 M NaHCO3 (3
ꢁ 10 mL), H2O (5 mL), 10% citric acid (4 ꢁ 10 mL), H2O (5 mL), and
brine (5 mL), dried (MgSO4), and filtered. The solvent was distilled off
in vacuo, and the residual oil was dried in vacuo over P2O5 at 30 ꢀC. This
oil self-solidified on storage at þ4 ꢀC and was recrystallized from
EtOAc/hexane to give 13 (2.61 g, 89%) as small colorless cubic crystals;
mp 80ꢀ81 ꢀC. TLC, NMR, and anal. C, H, N were conducted.
1-(N-Benzyloxycarbonyl)amino-8-[N-(tert-butyloxycarbonyl)]amino-
4-azanonane (14). A mixture of 13 (1.94 g, 5 mmol), 3-(N-benzylox-
ycarbonyl)amino-1-bromopropane (1.63 g, 6 mmol), and K2CO3
(2.07 g, 15 mmol) was stirred in DMF (20 mL) for 16 h at 45 ꢀC
followed by addition of K2CO3 (1.38 g, 10 mmol) and PhSH (0.88 g, 8
mmol). After stirring for additional 6 h at 20 ꢀC, the salts were separated
and washed with DMF, and combined DMF filtrates were evaporated
to dryness in vacuo. The residue was treated with a mixture of
EtOAc (25 mL) and H2O (15 mL), the water phase was extracted with
EtOAc (3 ꢁ 5 mL), and combined EtOAc extracts were washed with
H2O (5 mL) and brine (10 mL). Solvent was distilled off in vacuo, and
the residue was purified on a silica gel column (160 g) eluting
subsequently with CHCl3, CHCl3ꢀMeOH (100:1), and CHCl3ꢀ
MeOH (95:5), which after drying in vacuo over P2O5 at 30 ꢀC gave
14 (1.35 g, 69%) as a colorless oil. TLC, NMR, and anal. C, H, N were
conducted.
was dissolved in 2 M NaOH (5 mL) and extracted with DCM
(3 ꢁ 5 mL). Combined DCM extracts were washed with 1 M NaHCO3
(5 mL), H2O (3 mL), and brine (5 mL), dried (MgSO4), filtered, and
concentrated in vacuo. The residue was dried in vacuo over P2O5 to give
1-(N-benzyloxycarbonyl)amino-4-aza-8-aminononane free amine (0.62
g, 91%) as a viscous oil. Pd black in MeOH (∼0.5 mL) was added to a
solution of the above free amine (0.52 g, 1.77 mmol) in a mixture of
AcOHꢀMeOH (1:1, 6 mL total), and hydrogenation was carried out at
atmospheric pressure. The catalyst was filtered off, the residue was
washed with MeOH, and the combined filtrates were evaporated to
dryness in vacuo. The residue was dissolved in EtOH and upon addition
of HCl/EtOH (0.8 mL, 7 M) was evaporated to dryness in vacuo, and the
residue was recrystallized from MeOH/EtOH mixture to give 7a (0.4 g,
84.2%) as colorless crystals; mp 195ꢀ196 ꢀC; Rf 0.24 (C); Rf 0.12 (B).
1H NMR (D2O): δ 3.40 (1H, qdd, J = 8.6, 6.6, 4.9 Hz, CH); 3.20ꢀ3.07
(6H, m, 3*NCH2); 2.15ꢀ2.06 (2H, m, (NCH2)2CH2); 1.87ꢀ1.61 (4H,
m, CHCH2CH2); 1.32 (3H, d, J = 6.6 Hz, CH3). 13C NMR (D2O): δ
50.02, 49.93, 47.28, 39.33, 33.57, 26.46, 24.59, 20.04. Anal. C, H, N.
Cell Culture. The DU145 cell line was obtained from American
Type Culture Collection, USA. Mouse primary fetal fibroblasts were
obtained as described earlier.28 Cells were cultured in humidified
atmosphere at þ37 ꢀC, 10% CO2 in Dulbecco’s modified Eagle’s
medium supplemented with 10% heat-inactivated fetal bovine serum,
2 mM L-glutamine and 50 μg/mL gentamycin. The cells were lysed in a
buffer containing 50 mM potassium phosphate buffer, pH 7.2, 0.1 mM
EDTA, 0.1% Triton X-100, 0.1 mM dithiothreitol, and Complete
EDTA-free protease inhibitor (Roche Diagnostics). Aliquots of the
lysate were taken for polyamine measurement, and the rest was
centrifuged at 13 000 rpm for 20 min at þ4 ꢀC. The supernatant was
used for enzyme assays. Uptake competition experiments were pre-
formed as described earlier.25
Cytotoxicity Assay. DU145 cells were grown on 96-well plates
with 10 or 100 μM analogs in the presence or absence of 1 mM AG. After
72 h, 10 μL of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (5 mg/mL in PBS) was added, and cells were incubated for 4 h
before extracting the stain with a solution containing 4 mM HCl, 0.1%
Triton X-100, and 80% isopropanol. Absorbances were measured at
595 nm using a microplate reader.
Recombinant Enzyme Assays. Plasmids coding human SMO or
APAO were a kind gift from Dr. C. Porter, Roswell Park Cancer
Institute, New York, USA. Cloning of mouse recombinant SSAT was
described earlier.29 Recombinant proteins were produced and assays
were carried out as published previously.30
Analytical Methods. Alternative splicing of SSAT was analyzed as
described earlier.24 Polyamines were measured with HPLC according to
the published method.27 Acid-precipitated pellets were dissolved in 0.1
M NaOH, and the amount of DNA was measured using the PicoGreen
reagent (Invitrogen) according to manufacturer’s instructions. Enzyme
activities were measured from the cytosolic fractions as described
earlier.14,25,31,32
Statistical Analysis. One-way analysis of variance with Tuckey’s
post-hoc test was used for multiple comparisons with the aid of GraphPad
Prism 4.03 (GraphPad Software Inc.).
1,8-Diamino-4-azanonane Trihydrochloride, 8-MeSpd (7a). To a
cooled (0 ꢀC) solution of 14 (1.1 g, 2.8 mmol) in MeOH (7 mL) was
added HCl/MeOH (1.2 mL, 10 M), and the mixture was stirred for 30
min at 20 ꢀC followed by evaporation to dryness in vacuo (bath
temperature 20 ꢀC). The residue was dissolved in EtOH (2 mL) and
precipitated with an excess of Et2O, which after drying in vacuo over
P2O5/KOH resulted in 1-(N-benzyloxycarbonyl)amino-4-aza-8-amino-
nonane dihydrochloride (0.88 g, 85%); mp 134ꢀ136 ꢀC. TLC, NMR,
and anal. C, H, N were conducted. Thus obtained 1-(N-benzyloxycar-
bonyl)amino-4-aza-8-aminononane dihydrochloride (0.85 g, 2.3 mmol)
’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis and spectroscopic
b
details for 3-(N-benzyloxycarbonyl)amino-1-bromopropane,
physicochemical data for compounds 9a, 9b, 11, 12, 13, 14,
and 1-(N-benzyloxycarbonyl)amino-4-aza-8-aminononane dihy-
drochloride, elementary analysis results of target compounds and
table of metabolism and efflux of methylated analogs in DU145
cells. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Journal of Medicinal Chemistry
ARTICLE
’ AUTHOR INFORMATION
regeneration in transgenic rats with activated polyamine catabolism.
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(12) Hyv€onen, M. T.; Kein€anen, T. A.; Cerrada-Gimenez, M.;
Sinervirta, R.; Grigorenko, N.; Khomutov, A. R.; Veps€al€ainen, J.;
Alhonen, L.; J€anne, J. Role of hypusinated eukaryotic translation
initiation factor 5A in polyamine depletion-induced cytostasis. J. Biol.
Chem. 2007, 282, 34700–34706.
(13) Chattopadhyay, M. K.; Park, M. H.; Tabor, H. Hypusine
modification for growth is the major function of spermidine in Sacchar-
omyces cerevisiae polyamine auxotrophs grown in limiting spermidine.
Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 6554–6559.
(14) Hyv€onen, M. T.; Howard, M. T.; Anderson, C. B.; Grigorenko,
N.; Khomutov, A. R.; Veps€al€ainen, J.; Alhonen, L.; J€anne, J.; Kein€anen,
T. A. Divergent regulation of the key enzymes of polyamine metabolism
by chiral alpha-methylated polyamine analogs. Biochem. J. 2009,
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Khomutov, A. R.; Veps€al€ainen, J. J.; Sinervirta, R. M.; Kein€anen, T. A.;
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Corresponding Author
*E-mail: mervi.hyvonen@uef.fi. Tel. þ358403552092.
Author Contributions
§These authors contributed equally to this work.
’ ACKNOWLEDGMENT
The authors thank Ms. S. Juutinen, Ms. A. Karppinen, Ms. A.
Korhonen, Ms. T. Reponen, and Ms. M. Salminkoski for their
skillful technical assistance. The authors also thank Dr. R. A.
Casero, Jr. (Johns Hopkins University School of Medicine,
Baltimore, U.S.A), for critical reading of the manuscript. This
work was supported by grants from the Academy of Finland,
Russian Foundation for Basic Research (Project Nos. 09-04-
01272 and 08-04-91777), program Molecular and Cell Biology of
the Presidium of Russian Academy of Sciences, North Savo
Regional Fund of Finnish Cultural Foundation, and University of
Eastern Finland.
’ ABBREVIATIONS
AdoMetDC, S-adenosyl-L-methionine decarboxylase; APAO,
acetylpolyamine oxidase; dc-SAM, decarboxylated S-adenosyl-L-
methionine; DFMO, R-difluoromethylornithine (2,5-diamino-
2-(difluoromethyl)pentanoic acid); DENSpm, N1,N11-diethyl-
norspermine; N1-AcSpd, N1-acetylspermidine; N1-AcSpm, N1-
acetylspermine; NMD, nonsense-mediated mRNA decay; OAZ,
antizyme; ODC, ornithine decarboxylase; Put, putrescine (1,4-
diaminobutane); SAM, S-adenosyl-L-methionine; Spd, spermi-
dine (1,8-diamino-4-azaoctane); Spm, spermine (1,12-diamino-
4,9-diazadodecane); SMO, spermine oxidase; SSAT, spermi-
dine/spermine N1-acetyltransferase
(20) Seiler, N.; Delcros, J. G.; Moulinoux, J. P. Polyamine transport
in mammalian cells. An update. Int. J. Biochem. Cell Biol. 1996, 28,
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substrates of spermidine synthase. Int. J. Biochem. 1987, 19, 1037–1047.
(22) Kahana, C. Regulation of cellular polyamine levels and cellular
proliferation by antizyme and antizyme inhibitor. Essays Biochem. 2009,
46, 47–61.
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