Full text of DOI:10.1016/j.bmc.2007.07.030

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7426–7433
Construction of polyamine-modified uridine and adenosine
derivatives—evaluation of DNA binding capacity
and cytotoxicity in vitro
Johannes Ghatnekar,a Margareta Hagerlof,b Stina Oredsson,c Kersti Alm,c
¨
¨
Sofi K. C. Elmrothb,* and Tina Perssona,
aOrganic Chemistry, Chemical Center, Lund Univesity, PO Box 124, SE-221 00 Lund, Sweden
bBiochemistry, Chemical Center, Lund University, PO Box 124, SE-221 00 Lund, Sweden
cCell and Organism Biology, Lund University, Helgonav. 3B, SE-223 62 Lund, Sweden
Received 30 April 2007; revised 5 July 2007; accepted 10 July 2007
Available online 21 August 2007
Abstract—We here report the synthesis of the two polyamine-based nucleoside derivatives 5-{[bis-(3-aminopropyl)amino]acetam-
ido-1-propynyl}uridine and 2-{[bis-(3-aminopropyl)amino]-acetamido-1-propynyl}adenosine. The various polyamine derivatives
have been used in thermal melting analysis using DNA from herring testes, and in cellular studies using four different cell lines.
The compounds were all found to be non-toxic, thus holding good promise for future use as siRNA building blocks.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
level in the cellular machinery.8 Thus, development of
polyamine derivatives is of interest for future design of
polyamine-based drugs acting on RNA level. Because
of their simple structure and multiple roles, further stud-
ies of analogues of the natural polyamines are impor-
tant, both from a medicinal chemistry perspective and
to obtain a more detailed fundamental understanding
of their mode of action in vivo. In the latter context,
modified polyamine derivatives are of particular interest
for potential use as inhibitors of the endogenous poly-
amine biosynthetic pathway. In this study, the synthesis
and activity of a series of novel polyamine analogues is
reported. The polyamine structure has been altered by
chemical synthesis, and the analogues have been studied
with respect to their affinity to DNA, their influence on
DNA structure, and evaluated with respect to their
growth inhibitory activity in four different breast cancer
cell lines.9
Polyamines are small aliphatic organic molecules with
positive character that are present in prokaryotic and
eukaryotic cells.1 The naturally occurring polyamines
putrescine, spermidine and spermine are involved in
multiple cellular functions.2,3 Many studies support that
polyamines are involved in cell growth and differentia-
tion,4,5 but the mechanisms of action are still poorly
understood. Polyamines are also involved in chromatin
condensation, maintenance of DNA and RNA struc-
ture, RNA processing, translation, and protein activa-
tion.6,7 Recently, functionalized polyamines have been
shown to bind specifically to structured RNA indicating
that polyamines might play a regulatory role on RNA
Abbreviations: t-Boc, tert-butylcarbamate; DCC, N,N-Dicyclohexyl-
carbodiimide; DIEA, diisopropylethylamine; TFA, trifluoroacetic acid;
HCl, hydrochloric acid; DCM, dichloromethane; Et3N, triethylamine;
DMF, dimethylformamide; PFP, pentafluorophenol; MeOH,
methanol; RP C-18, reversed-phase C-18.
2. Results and discussion
2.1. Chemical synthesis
Keywords: Modified nucleoside; Adenosine; Uridine; Polyamine
derivatives.
*
Corresponding author. Tel.: +46 46 222 3692; fax: +46 46 222
4116; e-mail addresses: sofi.elmroth@biochemistry.lu.se;tina.persson@
organic.lu.se
The polyamine group was connected to the nucleoside
via linker to the C-5 position in the heterocyclic moiety
of uridine and to the C-2 position in the heterocyclic
Tel.: +46 46 222 8210; fax: +46 46 222 8209.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.030

J. Ghatnekar et al. / Bioorg. Med. Chem. 15 (2007) 7426–7433
7427
moiety of adenosine, respectively.10 In the first step, the
polyamine linker was functionalized with a triple bond
enabling Pd-catalysed coupling reactions to 5-iodouri-
dine and 2-iodoadenosine, respectively, using the Sono-
gashira reaction.11 The starting material 5-iodouridine
was commercially available, but the corresponding 2-
iodoadenosine was synthesized in four steps according
to the literature.12–14 In the first step, toward polyamine
derivative 7 (Scheme 1), the primary amine functions in
the commercially available polyamine 3,30-diaminodi-
propylamine 1 were protected using t-Boc groups15,16
producing compound 2 in 84% yield. The t-Boc groups
were used as they are stable toward hydrogenation
and basic reaction conditions. In the next step, benzyl
bromo acetate was used as alkylation reagent in the
presence of DIEA using DMF as solvent. Purification
of 3 by silica gel column chromatography using
EtOAc/heptane (70:30) as eluent produced white crys-
tals in 97% yield. The benzyl protecting group was re-
moved by hydrogenation at 60 psi using MeOH as
solvent.17,18 After evaporation, a white foam was
formed and polyamine derivative 4 was obtained in
90% yield. Formation of the carboxylic acid is essential
in order to generate the desired amide bond between the
N-propargylic amine and the polyamine derivative. For
our purpose, the amide bond was chosen since it is more
stable toward hydrolysis in aqueous media. Prior to con-
necting N-propargylamine, the carboxylic acid was acti-
vated by reacting with pentafluorphenol in the presence
of DCC.19 The reaction mixture was stirred overnight,
whereupon it was filtered through a Celite pad. The
resulting mother liquid was evaporated to dryness and
the crude product 5 was used in the next step without
further purification. The activated polyamine derivative
5 was reacted with N-propargylamine in the presence of
DIEA, and stirred under an inert atmosphere over-
night.11 The product was purified by silica gel column
chromatography using EtOAc/Heptane (80:20) as eluent
producing 6 in 61% yield. Deprotection of polyamine
derivatives 4 and 6 was accomplished by using TFA in
DCM giving the deprotected polyamine derivatives 4d
and 7 in 95% yields. The polyamine derivative 6 was suc-
cessfully connected to 5-iodouridine 8 and 2-iodoadeno-
sine 11, respectively, by using Sonogashira reaction
conditions (Scheme 2).
Nucleoside derivatives 8 and 11 were dissolved in dry
DMF, treated with CuI, Pd(PPh3)4 and reacted with 6
overnight at room temperature. The corresponding
polyamine-based nucleoside analogues 9 and 12 were
purified by silica gel column chromatography using a
mixture of DCM/MeOH (9:1) and DCM/MeOH
(98:2–9:1), respectively, as eluent. The Pd-cross coupling
reaction between the polyamine derivative 6 and 5-iodo-
uridine 8 or 2-iodoadenosine 11 resulted in 95 and 51%
yields of 9 and 12, respectively.
Removal of the t-Boc groups was first performed with
the uridine derivative 9 using TFA and anisole as re-
agents. The resulting oil was dissolved in water and
washed with DCM, whereupon the aqueous phase was
evaporated to dryness under reduced pressure. The
product 10 was obtained as a brown oil in a yield of
about 25%. Exposure of the corresponding adenosine
derivative 12 to TFA and anisole resulted in a brown
syrup-like mixture which was difficult to dissolve in
water. Various attempts to separate the produced prod-
uct mixture failed. Therefore, an alternative approach
was considered with the aim to couple the deprotected
polyamine derivative 7 to 2-iodoadenosine 11. By using
the above described Sonogashira reaction conditions,
the polyamine-based adenosine analogue 13 was ob-
tained in 19% yield. To simplify the purification proce-
dure, a slight excess of 11 over the deprotected
polyamine derivative 7 was used and after RP C-18 col-
umn chromatography using MeOH as eluent. Analysis
of 1H NMR indicated correct product formation and re-
moval of impurities. Further purification was accom-
BocHN
N
NHBoc
a
c
b
O
H2N
N
H
NH2
BocHN
N
H
NHBoc
O
3
1
2
d
e
f
BocHN
N
HO
f
NHBoc
BocHN
N
NHBoc
BocHN
N
NHBoc
H2N
N
NH2
4
5
6
7
O
O
F
O
O
O
F
HN
HN
F
F
F
H2N
N
NH2
O
4d
HO
Scheme 1. Reagents and conditions: (a) 1, tert-butyl phenyl carbonate, DMF, rt, 12 h, 84%; (b) 2, benzyl bromoacetate, DMF, DIEA, rt, 4 h, 97%;
(c) 3, Pd/C 10%, MeOH, 60 psi, rt, 22 h, 90%; (d) 4, solution; i—pentafluorophenol, EtOAc, solution; ii—DCC, EtOAc; combine (i) and (ii) and react
at rt; used in the next step without purification; (e) 5, solution; i—DIEA, DCM solution; ii—N-propargylamine, DCM, combine (i) and (ii) and react
at rt, 61%; (f) 6, TFA, DCM, rt, 95%.

7428
J. Ghatnekar et al. / Bioorg. Med. Chem. 15 (2007) 7426–7433
Scheme 2. Reagents and conditions: (a) 6 or 7, Pd(PPh3)4, CuI, DMF, Et3N, rt; (b) 9, TFA, anisole, MeOH, rt, 10%.
plished by using RP C-18 column chromatography and
H2O as eluent producing the polyamine-based analogue
13 as a yellow oil in 19% yield.
12 ꢁC to 77 ꢁC as expected from theory and previous re-
ports.21 In the presence of 4d and 7, the Tm was however
decreased (Fig. 1). The effect can be ascribed to the pres-
ence of the tertiary substituted central N-atom of 4 and
7 which introduces more steric constrains on the interac-
tion with DNA. The electron density at the central N is
also changed. In contrast, the modified nucleosides 10
and 13 did not have any effect on the melting tempera-
ture (Table 1). However, a careful inspection of the
melting curves obtained for these two latter compounds
shows a slightly altered profile above Tm, indicating a
slower transition from duplex to single-stranded coil
for these compounds, see Figure 1. Only minor pH
changes of the solution were observed after addition of
the polyamines, for example, an increase of 0.06 pH
units after addition of 1 and decrease of 0.08 and 0.14
units for 4d and 7, respectively.
2.2. Thermal melting analysis
The thermal melting point (Tm) of DNA from herring
testes (Sigma) was determined to be 65 ꢁC in 10 mM
NaCl, which is comparable to results obtained by oth-
ers.20 Thermal melting studies were further performed
with DNA from herring testes in the presence of poly-
amines 1, 4d, 7, 10 and 13, see Schemes 1 and 2 for illus-
trations. In the presence of 1, the Tm was increased by
1.3
1.2
1.1
1
Table 1. Thermal melting points for herring testes DNA in the
presence of the polyamines 1, 4d, 7, 10 and 13
Polyamine
Conc (lM)
Tm (ꢁC)
DTm (ꢁC)
—
1
—
10
1
65
77
62
61
50
58
52
51
66.5
66
—
12
4d
ꢀ3
ꢀ4
ꢀ15
ꢀ7
ꢀ13
ꢀ14
1.5
1
10
100
10
50
100
10
10
25
45
65
85
7
Temperature (˚C)
Figure 1. Absorbance versus temperature profiles of herring testes
DNA alone (s) and in the presence of 1 (h), 4d (d), 7 (j), 10 (Ç) and
13 (m). The DNA concentration was kept to 100 lM, and the
concentration of the polyamines was 10 lM. The measurements were
performed in 10 mM NaCl.
10
13
The concentration of DNA was typically 100 lM and the measure-
ments were performed in 10 mM NaCl.

J. Ghatnekar et al. / Bioorg. Med. Chem. 15 (2007) 7426–7433
7429
2.3. Circular dichroism
while even stimulation of MTT reduction was found in
MCF-7 cells.
The influence of compounds 1, 4d, 7, 10, and 13 on the
circular dichroism (CD) spectra of B-DNA22 is shown
in Figure S1. After addition of the polyamines, the
spectra show the characteristics of B-DNA, that is, a
positive peak at 275 nm, a cross-over at 258 nm, and
a negative peak at 246 nm. The polyamine with the
largest effect on DNA is 1, which induces both a higher
peak at 275 nm and an elevated minimum at 246 nm.
Polyamines 4d and 13 induce similar spectral changes
as does 1, while the effect of 7 and 10 are slightly less
pronounced.
3. Conclusions
In summary, we synthesized two new polyamine-based
nucleoside derivatives. Both nucleoside derivatives and
their corresponding polyamine precursors were shown
to have a limited influence on DNA structure and ther-
mal stability. In comparison to the unmodified 3,30-
diaminodipropylamine 1, the polyamine derivatives 4d
and 7 were found to have a slight destabilizing effect
on the DNA structure, whereas the nucleoside deriva-
tives 10 and 13 had little effect. None of the polyamine
derivatives showed any toxic effect in the breast cancer
cell lines. The compounds thus seem to be well suited
for further work aiming at synthetic incorporation of
these bases into, e.g., modified siRNAs, since the toxic
side effects arising from tentative degradation products
are likely to be minimal.
2.4. Cellular studies
In the MTT assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl tetrazolium bromide (MTT) is added to the med-
ium of the cells in the microwell. After removal of the
MTT containing medium, the formazan crystals formed
were dissolved by addition of dimethyl sulfoxide
(DMSO) and the absorbance was monitored. The
amount of formazan generated is assumed to be directly
proportional to the cell number when using homoge-
nous cell populations.23–25
4. Experimental
4.1. General information
The toxicity of 4d, 7, 10 and 13 on four human breast
cancer cell lines is shown in Figure 2.
1H NMR was recorded on a Bruker DRX400 spectrom-
eter operating at 400 MHz for H and 100.6 MHz for
1
As can be seen here, none of the compounds exhibited
strong cytotoxicity in any of the cell lines studied. The
figure also shows that there are no clear patterns in
the response to the compounds. However, the general
conclusion may be drawn that HCC1937 and L56Br-
C1 show a more similar response than the other two cell
lines. SK-BR-3 cells appear to be the least responsive,
13C. A Sanyo Gallenkamp melting point apparatus
was used for determination of the melting points of
the polyamine derivatives. All melting points are uncor-
rected. IR spectra were recorded on a Nicolet Avastar
360 FT-IR ESP. TLC was carried out on alumina plates
covered with silica (Merck 60 F254). Ninhydrin 1% was
used for visualization of polyamine derivatives. Column
Figure 2. Effect of compounds 4d (d), 7 (j), 10 (Ç) and 13 (m) on four human breast cancer cell lines evaluated with a MTT assay. Cells were seeded
in 96-well plates and incubated for 24 h before addition of the compounds to the final concentrations (shown in the figure). After 48 h of treatment,
the effects were evaluated using MTT assay. Each data point is the mean value of 12–18 independent wells from 2 to 3 independent experiments.

7430
J. Ghatnekar et al. / Bioorg. Med. Chem. 15 (2007) 7426–7433
˚
chromatography was performed on silica 60 A (0.35–
0.70) and silica C18—reversed phase, 17% C (40–
63 lm). Celite 545 was used as media in various filtra-
tion steps. All solvents were of PA quality and prior
duced. Compound 4 was obtained in a yield of 1.2 g
(90%); IR (KBr, cmꢀ1): 1696 (C@O); 1H NMR
(400 MHz, CD3OD, ppm): d 1.44 (s, 18H), 1.87 (m,
J = 7.6, 7.1, 6.3 Hz, 4H), 3.17 (m, J = 6.6 Hz, 8H),
3.62 (s, 2H); 13C NMR (100.6 MHz, CD3OD, ppm): d
26.2, 28.9, 38.3, 54.7, 57.0, 80.5, 158.9, 170.2; HRMS
(FAB+) m/z calcd for C18H35O6N3: 389.2526. Found:
390.2605 (M+H)+. Compound 4d, used for cellular,
thermal, melting, and CD studies, was obtained after
addition of TFA to 4 as described below (Section 4.7).
˚
to use dried over molecular sieves 4 A. Chemicals and
solvents were purchased from Aldrich and Acros.
DNA from herring testes was purchased from Sigma,
and the concentration was determined spectroscopically
using the average molecular extinction coefficient per
26
DNA base, e260 = 6521 Mꢀ1 cmꢀ1
.
3,30-Diaminodi-
propylamine 1 was purchased from Aldrich. Concentra-
tions of 4d and 7 were determined by use of molecular
weights of 186.12 g molꢀ1 for 4d and 262.32 g molꢀ1
for 7, respectively.
4.5. Synthesis of [Bis-(3-tert-butoxycarbonyl-aminopro-
pyl)amino]acetic acid 2,3,4,5,6-pentafluorophenyl ester (5)
Compound 4 (1.2 g, 3.2 mmol) and pentafluorophenol
(0.77 g, 4.2 mmol) were dissolved in 11 ml dry EtOAc.
In a second flask, DCC (890 mg, 4.3 mmol) was dis-
solved in 2 ml EtOAc. Each flask was cooled on ice bath
while stirring under an argon atmosphere. The two solu-
tions were combined, the reaction mixture was stirred
overnight and the reaction temperature was allowed to
slowly reach room temperature. The resulting reaction
mixture was cooled on an ice bath and filtered through
a Celite pad and carefully washed with EtOAc. The
mother liquid was evaporated resulting in a clear viscous
oil. The product was used in the next step without fur-
ther purification and analysis.
4.2. Synthesis of Bis-(3-tert-butoxycarbonyl-aminopro-
pyl)amine (2)
Compound 2 was synthesized according to the literature
procedure. The isolated product was obtained as white
crystals in a yield of 84% (8.0 g). Analysis of the product
was made according to the literature.15
4.3. Synthesis of [Bis-(3-tert-butoxycarbonyl-aminopro-
pyl)amino]acetic acid benzylester (3)
Compound 2 (4.0 g, 12 mmol) and 9 ml DMF were dis-
solved in a three-necked flask under stirring. The mix-
ture was cooled by using an ice bath followed by
addition of benzylbromo acetate (2.4 ml, 15 mmol) and
DIEA (2.1 ml, 12 mmol) over a time period of 30 min.
The resulting reaction mixture was stirred at 0 ꢁC for
additional 30 min, whereupon it was allowed to reach
rt. The reaction mixture was reacted for additional 4 h
at rt. Ether (50 ml) was added and the formed white pre-
cipitate was collected by filtration and the mother liquid
was evaporated to dryness. The produced oil was dis-
solved in 50 ml DCM and carefully washed sequentially
with 3 · 40 ml water, 2 · 40 ml 1 M HCl, and 3 · 40 ml
water. The organic phase was dried over Na2SO4 fol-
lowed by evaporation to dryness. The crude product
was purified by silica gel column chromatography using
EtOAc/Heptane (70:30) as eluent and 3 was obtained in
a yield of 5.6 g (97%); IR (KBr, cmꢀ1): 1733 (C@O); 1H
NMR (400 MHz, CDCl3, ppm): d 1.43 (s, 18H), 1.60 (m,
J = 6.4, 6.3 Hz, 4H), 2.57 (t, J = 6.5 Hz, 4H), 3.17 (m,
J = 5.9 Hz, 4H), 3.32 (s, 2H), 5.15 (s, 2H), 5.28 (bs,
2H), 7.34 (m, 5H); 13C NMR (100.6 MHz, CDCl3,
ppm): d 27.5, 28.7, 40.0, 52.3, 55.1, 66.6, 79.1, 128.6,
128.8, 135.8, 156.3, 171.4; M.p. 69.9-71.5ꢁC; HRMS
(FAB+) m/z calcd for C25H41O6N3: 479.2995. Found:
480.3071 (M+H)+.
4.6. Synthesis of [Bis-(3-tert-butoxycarbonyl-aminopro-
pyl)amino]acetamido-1-propynyl (6)
Compound 5 (2.3 g, 4.1 mmol) and DIEA (1.4 ml,
8.2 mmol) were dissolved in 13 ml DCM. In parallel, a
solution of N-propargylamine (0.53 ml, 8.2 mmol) was
dissolved in 3.5 ml DCM. The two solutions were mixed
and reacted for 12 h at rt under argon atmosphere. The
resulting reaction mixture was concentrated under re-
duced pressure, and the crude product was purified by
silica gel column chromatography using EtOAc/Hep-
tane (80:20) as eluent. The product was obtained as a
red oil in a yield of 61% (906 mg); IR (NaCl, cmꢀ1):
2114; 1H NMR (400 MHz, CDCl3, ppm): d 1.41 (s,
18H), 1.59 (m, J = 6.9 Hz, 4H), 2.20 (t, J= 2.4 Hz,
1H), 2.47 (t, J = 7 Hz, 4H), 3.00 (s, 2H), 3.14 (m,
J = 6.2 Hz, 4H), 4.04 (dd, J = 3.2, 2.5 Hz, 2H), 4.87
(br s, 2H), 7.59 (br s, 1H); 13C NMR (100.6 MHz,
CDCl3, ppm): d 27.8, 28.6, 28.7, 38.5, 50.7, 58.7, 71.3,
79.4, 79.9, 156.3, 171.6; HRMS (FAB+) m/z calcd for
C21H38O5N4: 426.2842. Found: 427.2933 (M+H)+.
4.7. Synthesis of [Bis-(3-aminopropyl)amino]acetamido-1-
propynyl (7)
4.4. Synthesis of [Bis-(3-tert-butoxycarbonyl-aminopro-
pyl)amino]acetic acid (4)
TFA (2.2 ml, 28.6 mmol) was slowly added to a flask
containing 6 (497 mg, 1.16 mmol) dissolved in 10 ml
dry DCM, whereupon the mixture was stirred overnight
at rt. The reaction mixture was evaporated to dryness
under reduced pressure resulting in a brown oil in a yield
of 95% (249 mg); IR (KBr, cmꢀ1): 2131; 1H NMR
(400 MHz, CD3OD, ppm): d 1.85 (m, J = 6.9 Hz, 4H),
2.63 (t, J = 2.5 Hz, 1H), 2.67 (t, J = 6.8 Hz, 4H), 3.02
(t, J = 7.0, 4H), 3.28 (s, 2H), 4.01 (d, J = 2.5 Hz, 2H);
13C NMR (100.6 MHz, CD3OD, ppm): d 27.8, 29.9,
Compound 3 (1.7 g, 3,5 mmol), Pd/C 10% (262 mg,
15 wt%), and 80 ml MeOH were mixed in a sealed tube,
whereupon hydrogen pressure of 60 psi was applied. The
mixture was reacted for 22 h at rt. The resulting reaction
mixture was filtered through a Celite pad and carefully
washed with 100 ml MeOH. The mother liquid was
evaporated to dryness and a white product was pro-

J. Ghatnekar et al. / Bioorg. Med. Chem. 15 (2007) 7426–7433
7431
37.9, 53.7, 55.5, 73.1, 79.8, 165.8; HRMS (FAB+) m/z
calcd for C11H22ON4: 226.1794. Found: 227.1881
(M+H)+.
reduced pressure. The obtained oil was dissolved in
water and extracted with 3 · 5 ml DCM. The aqueous
phase was evaporated to dryness and the produced
brown oil was dissolved in MeOH, 1 ml DCM, where-
upon about 110 mg Dowex 1 · 8 ꢀ 200 was added to
the solution. The mixture was stirred for additional
30 min followed by filtration and evaporation to dry-
ness. The resulting product was obtained in 25% yield;
1H NMR (400 MHz, CD3OD, ppm): d 1.70 (m,
J = 6.8, 6.7 Hz, 4H), 2.55 (t, J = 6.9 Hz, 4H), 2.77 (t,
J = 6.7, Hz, 4H), 3.13 (s, 2H), 3.74 (dd, J = 9.4,
2.9, Hz, 1H), 3.86 (dd, J = 9.9, 2.5 Hz, 1H), 4.00 (m,
1H), 4.14 (m, J = 3.2 Hz, 2H), 4.20 (s, 2H), 5.89 (bs,
1H), 8.23 (s, 1H); 13C NMR (100.6 MHz, CD3OD,
ppm): d 24.5, 30.6, 38.8, 53.7, 56.7, 62.1, 71.2, 75.5,
76.2, 86.7, 89.8, 91.1, 100.0, 145.7, 151.5, 164.6,
174.1; HRMS (FAB+) m/z calcd for C20H32O7N6:
468.2332. Found: 469.2405 (M+H)+.
4.8. Synthesis of 5-{[Bis-(3-tert-butoxycarbonyl-amino-
propyl)amino]acetamido-1-propynyl}uridine (9)
Compound 8 (159 mg, 0.43 mmol), CuI (28.6 mg,
0.15 mmol), Pd(PPh3)4 (48.5 mg, 42 lmol), and com-
pound 6 (526 mg, 1.23 mmol) were dissolved in 1 ml
DMF and Et3N (114 ll, 0.82 mmol). After dissolution,
4 ml DMF was added and the reaction was stirred under
argon atmosphere for 22 h at rt. The reaction mixture
was concentrated under reduced pressure, and the crude
product was purified by silica gel column chromatogra-
phy using DCM/MeOH (9:1) as eluent. The product was
isolated as yellow crystals in a yield of 95% (273 mg); IR
(KBr, cmꢀ1): 3062; 1H NMR (400 MHz, CD3OD, ppm):
d 1.43 (s, 18H), 1.64 (dd, J = 6.9, 6.6 Hz, 4H), 2.53 (t,
J = 7.1, 6.8 Hz, 4H), 3.10 (t, J = 7.1, 6.6 Hz, 6H), 3.75
(dd, J = 9.5, 2.7 Hz, 1H), 3.88 (dd, J = 9.6, 2.6 Hz,
1H), 4.00 (m, 1H), 4.16 (m, 2H), 4.20 (s, 2H), 5.88 (d,
J = 3.6 Hz, 1H), 8.37 (s, 1H); 13C NMR (100.6 MHz,
CD3OD, ppm): d 28.6, 29.1, 30.2, 39.5, 53.9, 59.3,
62.1, 71.2, 75.1, 76.2, 80.1, 86.6, 90.4, 91.1, 100.2,
145.7, 151.6, 158.7, 164.6, 174.3; mp 82.1–88.1 ꢁC;
HRMS (FAB+) m/z calcd for C30H48O11N6: 668.3381.
Found: 669.3483 (M+H)+.
4.11. Synthesis of 2-{[Bis-(3-aminopropyl)amino]acetam-
ido-1-propynyl}adenosine (13)
Compound 11 (150 mg, 0.38 mmol), CuI (24 mg,
0.13 mmol), Pd(PPh3)4 (44 mg, 38 lmol) were dissolved
in 1 ml DMF and Et3N (105 ll, 0.75 lmol). Compound
7 dissolved in 4 ml DMF was added to the mixture and
the resulting solution was stirred overnight under an ar-
gon atmosphere. The solvent was removed by reduced
pressure and the resulting brown oil was dissolved in
water and purified by reversed-phase column chroma-
tography, C-18, using MeOH as eluent. A second puri-
fication was accomplished made on C-18 column using
H2O as eluent. The product was isolated as yellow oil
in a yield of 19% (35.0 mg); 1H NMR (400 MHz,
CD3OD, ppm): d 1.88 (m, J = 6.7, 6.5 Hz, 4H), 2.66 (t,
J = 6.5 Hz, 4H), 3.06 (t, J = 6.8 Hz, 4H), 3.28 (s, 2H),
3.75 (dd, J = 10.0, 2.5 Hz, 1H), 3.87 (dd, J = 10.2, 2.3,
2.0, Hz, 1H), 4.17 (m, J = 2.3 Hz, 1H), 4.27 (m,
J = 2.5, 2.4 Hz, 2H), 4.33 (t, J = 2.4, 2.5 Hz, 1H), 4.68
(t, J = 5.8, 5.5 Hz, 1H), 6.96 (d, J = 6.3 Hz, 1H), 8.33
(s, 1H); 13C NMR (100.6 MHz, CD3OD, ppm): d 25.5,
30.0, 39.8, 53.6, 58.0, 63.6, 72.7, 75.8, 82.2, 83.3, 88.3,
91.0, 116.9, 142.8, 146.9, 150.2, 157.4, 173.8; HRMS
(FAB+) m/z calcd for C21H33O5N9: 491.2605. Found:
492.2693 (M+H)+.
4.9. Synthesis of 2-{[Bis-(3-tert-butoxycarbonyl-amino-
propyl)amino]acetamido-1-propynyl}adenosine (12)
Compound 11 (152 mg, 0.39 mmol), CuI (25 mg, 0.13
mmol), Pd(PPh3)4 (45 mg, 0.039 mmol), and compound
6 (496 mg, 1.16 mmol) were dissolved in 1 ml DMF and
Et3N (107 ll, 0.77 mmol). After dissolution, 4 ml DMF
was added and the reaction mixture was stirred at rt for
17 h under argon atmosphere. The produced mixture
was evaporated to dryness and the crude product was
purified by silica gel column chromatography using a
gradient of DCM/MeOH (98:2–9:1) as eluent. The iso-
lated product was obtained in a yield of 51% (136 mg);
1
IR (KBr, cmꢀ1): 2246; H NMR (400 MHz, CD3OD,
ppm): d 1.39 (s, 18H), 1.67 (dd, J = 7.1, 6.7 Hz, 4H),
2.56 (t, J = 7.0, Hz, 4H), 3.11 (t, J = 6.7 Hz, 4H), 3.14
(s, 2H), 3.75 (dd, J = 9.9, 2.7 Hz, 1H), 3.88 (dd,
J = 10.1, 2.5 Hz, 1H), 4.17 (q, J = 2.6, 2.5 Hz, 1H),
4.28 (s, 2H), 4.33 (q, J = 2.7, 2.3 Hz, 1H), 4.70 (t,
J = 6.0, 5.3 Hz, 1H), 5.93 (d, J = 6.2, Hz, 1H), 8.34 (s,
1H); 13C NMR (100.6 MHz, CD3OD, ppm): d 28.5,
28.9, 29.8, 39.5, 54.0, 59.3, 63.6, 72.6, 75.6, 80.1, 82.2,
83.5, 88.2, 91.3, 120.6, 142.9, 147.0, 150.2, 157.4,
158.7, 174.2; mp 92.2–102.6 ꢁC; HRMS (FAB+) m/z
calcd for C31H49O9N9: 691.3653. Found: 714.3559
(M+Na)+.
4.12. Melting experiments
Thermal melting profiles were obtained in 10 mM NaCl
to be directly comparable with previous investigations.20
Melting temperatures were determined for DNA from
herring testes alone, and herring testes DNA in the
presence of compounds 1, 4d, 7, 10 and 13. The concen-
tration of DNA was typically 100 lM and the concen-
tration of polyamine was 10 lM. Measurements were
performed using a Cary 300 Bio spectrophotometer at
260 nm. The temperature was increased at a rate of
0.4 ꢁC/min over a range of 25–98 ꢁC, allowing the sys-
tem to stabilize for 15 min before taking the first read-
ing. Readings were taken at every degree using a
signal average time of 2 s. The thermal melting point,
Tm, was determined from the peak of the first derivative
of the absorbance versus temperature curves (Figure
4.10. Synthesis of 5-{[Bis-(3-aminopropyl)amino]acetam-
ido-1-propynyl}uridine (10)
Compound
9
(54 mg, 80 mmol), TFA (300 ll,
4.0 mmol) and anisole (150 ll, 1.4 mmol) were dis-
solved in 4 ml dry MeOH. The reaction mixture was
stirred overnight at rt and evaporated to dryness under

7432
J. Ghatnekar et al. / Bioorg. Med. Chem. 15 (2007) 7426–7433
S2). The pH was measured before and after addition of
the polyamines.
of 0.055 · 106 cells per millilitre. A 180-ll aliquot of the
cell suspension was seeded per well in 96-well plates. After
24 h of incubation, the compounds to be tested were
added in 20 ll aliquots to the medium to give a final con-
centration of 0.1, 1, 10, or 100 lM. The compounds were
dissolved in H2O or PBS as 4 or 20 mM stock solutions.
The stock solutions were sterile-filtered and stored at
ꢀ20 ꢁC. Further dilutions before addition to the 96-well
plates were done in PBS. Controls received 20 ll PBS.
At 24, 48, and 72 h after addition of compounds, 20 ll
of a MTT solution was added to each well and the 96-well
plates were returned to the incubator for 1 h. The sterile-
filtered MTT solution (5 mg/ml in PBS) was stored pro-
tected from light at ꢀ20 ꢁC until usage. Then, the MTT
containing medium was removed and each well was gently
washed with PBS. The blue formazan product was dis-
solved by the addition of 100 ll of 100% DMSO per well.
The plates were swirled gently for 10 min to dissolve the
precipitate. Absorbance was monitored at 540 nm using
a Labsystems iEMS Reader MF (Labsystems Oy, Hel-
sinki, Finland) and the software DeltaSoft II v. 4.14 (Bio-
metallics Inc., Princeton, NJ, USA). The outermost wells
were not used for the calculations. The values were ex-
pressed as percentage of control one standard
deviation.
4.13. Circular dichroism
Circular dichroism measurements (CD) of DNA were
performed in the presence of the polyamines 1, 4d, 7,
10, and 13. The concentration of DNA was 100 lM
and the concentration of the polyamines was 10 lM.
The measurements were performed in 10 mM NaCl at
ambient temperature. All spectra were collected four
times consecutively on a Jasco J600 Spectropolarimeter
and an average was taken. Scans were obtained from
320 to 220 nm at a rate of 20 nm/min in a 3-ml cuvette
with a path length of 1 cm. The ellipticity given, h
(mdeg), was converted to the decadic molar extinction
coefficient, De, by first subtracting the base line and then
applying the following equation,
h
De ¼
ð1Þ
32:98 ꢁ cl
where c is the concentration in mol · dmꢀ3 and l is the
path length in cm.
4.14. Cellular experiments
4.14.1. Materials. Growth medium components were
purchased from Biochrom, Berlin, Germany. Tissue
culture plastics were purchased from Nunc, Roskilde,
Denmark. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tet-
razolium bromide (MTT) was purchased from ICN Bio-
medicals Inc., Aurora, OH, USA. Phosphate-buffered
saline (PBS: 8 g/l NaCl, 0.2 g/l KCl, 1.15 g/l Na2HPO4,
0.2 g/l KH2PO4, pH 7.3) was purchased from Oxoid
Ltd., Basingstoke, Hampshire, United Kingdom.
DMSO was purchased from Merck KGaA, Darmstadt,
Germany. The MCF-7 (HTB-22), HCC1937 (CRL-
2336), and SK-BR-3 (HTB-30) cell lines were purchased
from American Type Culture Collection, Manassas, VA,
USA. The L56Br-C1 cell line was established in Lund.9
Acknowledgments
¨
We are grateful for financial support from the FLAK
(Forskarskolan i La¨kemedelsvetenskap) at Lund Uni-
versity, Crafoordska Stiftelsen, The Royal Physiograph-
ical Society in Lund, The Gunnar Nilsson Foundation,
The Mrs. Berta Kamprad Foundation, The Swedish
Cancer Society (Contract No. 040607), and The Swedish
Research Council (Contract No. 40446101).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2007.07.030.
4.14.2. Cell culture experiments. All cell lines were cul-
tured at 37 ꢁC, in a humidified incubator with 5% CO2
in air. The human epithelial adenocarcionoma breast
cancer cell line MCF-7 was cultured in RPMI 1640 med-
ium supplemented with 10% fetal calf serum (FCS),
non-essential amino acids, insulin (10 lg/ml), penicillin
(50 U/ml), and streptomycin (50 mg/ml). The human
adenocarcinoma breast cancer cell line SK-BR-3 was
cultured in the same medium as MCF-7 cells, but with-
out the addition of insulin. The human carcinoma breast
cancer cell line HCC193727 was cultured in MEM a-
medium supplemented with 10% heat-inactivated FCS,
non-essential amino acids, gentamycin (0.1 mg/ml), epi-
dermal growth factor (EGF) (20 ng/ml) and insulin
(10 lg/ml). The human breast cancer cell line L56Br-
C1 was cultured in RPMI 1640 medium supplemented
with 10% heat-inactivated FCS, non-essential amino
acids, insulin (10 lg/ml), penicillin (50 U/ml), and strep-
tomycin (50 rmug/ml).
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