N-benzylpolyamines as vectors of boron and fluorine for cancer therapy and imaging: Synthesis and biological evaluation

Article abstract of DOI:10.1021/jm010897q

Cancer cells have high-affinity polyamine uptake systems with a low stringency for structural features. Putrescine, spermidine, and spermine have, therefore, been considered as potential vectors for the selective accumulation in tumors of therapeutically

Full text of DOI:10.1021/jm010897q

J . Med. Chem. 2001, 44, 3653-3664
3653
N-Ben zylp olya m in es a s Vector s of Bor on a n d F lu or in e for Ca n cer Th er a p y a n d
Im a gin g: Syn th esis a n d Biologica l Eva lu a tion
Be´ne´dicte Martin,† Franc¸oise Posse´me´,‡ Caroline Le Barbier,‡ Franc¸ois Carreaux,‡ Bertrand Carboni,‡
Nikolaus Seiler,† J acques-Philippe Moulinoux,† and J ean-Guy Delcros*,†
Groupe de Recherche en The´rapeutiques Anticance´reuses, Faculte´ de Me´decine, UPR ESA CNRS 6027, 2 Avenue du Pr. Le´on
Bernard, F35043 Rennes Cedex, France, and Synthe`se et Electrosynthe`se Organiques, Institut de Chimie, UMR CNRS 6510,
Avenue du Ge´ne´ral Leclerc, F35042 Rennes Cedex, France
Received April 9, 2001
Cancer cells have high-affinity polyamine uptake systems with a low stringency for structural
features. Putrescine, spermidine, and spermine have, therefore, been considered as potential
vectors for the selective accumulation in tumors of therapeutically or diagnostically useful
structures and elements. We envisaged N-benzyl derivatives of the polyamines as vectors of
10B and 18F for boron neutron capture therapy (BNCT) and tumor imaging by positron emission
tomography (PET), respectively. In the present work, the synthesis, transport characteristics,
DNA-binding properties, and cytotoxicity of several N-benzyl derivatives of putrescine and
spermidine are described. The fluorinated spermidine derivative N-{3-[(4-aminobutyl)amino]-
propyl}[(4-fluorophenyl)methyl]amine (N1-4-F bz-sp d ) may be useful for PET because of its
high accumulation in cancer cells via the polyamine transport system. Among the boron-
containing benzyl polyamines, N-(4-aminobutyl){[4-(dihydroxyboryl)phenyl]methyl}amine (4-
Bbz-p u t) and N-{3-[(4-aminobutyl)amino]propyl}{[4-(dihydroxyboryl)phenyl]methyl}amine (N1-
4-Bbz-sp d ) should be suitable for BNCT, because their accumulation in B16 melanoma cells
was more efficient than that of borocaptate and borophenylalanine, two reference compounds
used in BNCT.
In tr od u ction
inactivator of ornithine decarboxylase, greatly enhances
polyamine uptake by various cell lines.7-10 In vivo, the
polyamine content of tumor cells is more efficiently
depleted by DFMO than that of nongrowing or slow-
growing cells. As a consequence, polyamine depletion
increases the uptake rate of tumor cells, which readily
accumulate high amounts of polyamines.11-13
(iv) Another property of interest of spermidine and
spermine for tumor targeting is their high-affinity
binding to DNA.14 If a polyamine derivative is taken
up via the polyamine transport system, it will in part
be located in the cell nucleus.
All these properties have led many researchers to
design polyamine derivatives for tumor targeting. Known
chemotherapeutic drugs have been conjugated to poly-
amines to enhance cytotoxicity to tumor cells and to
diminish secondary effects on normal cells. For example,
DNA damage by a chlorambucil-spermidine conjugate
was enhanced,15 and among others, nitroimidazole16 and
aziridine17 polyamine conjugates have been synthesized,
with improved therapeutic indexes.
The polyamine transport system has also been used
to accumulate various isotopes in several cancer cells,
for either cancer curing or tumor imaging. Boron
neutron capture therapy (BNCT) is an emerging modal-
ity for the treatment of brain tumors and melanomas.
It relies on the formation of energy-rich R particles with
a short action radius by irradiation of the 10B isotope
with low-energy neutrons (10B(n,R)7Li); for reviews, see
refs 18 and 19. o-Carborane cages attached to the
primary or secondary amino groups of spermidine and
spermine have been made.20,21 In vitro, these compounds
bind to DNA and accumulate in tumor cells similarly
Putrescine, spermidine, and spermine are ubiquitous
low molecular weight polycations, essential for cell
growth and differentiation.1 In eukaryotic cells complex,
highly regulated biosynthetic and catabolic pathways
determine the intracellular pools of the polyamines.
Cells are also equipped with specific, active transport
systems, allowing them to take up polyamines from the
environment. Uptake and release are also essential for
pool-size control. The activity of the uptake system is,
among others, under the control of the intracellular free
polyamines. Structural features, and the enhanced
requirement of tumor cells for the polyamines, together
with the characteristics of the transport systems make
the polyamines attractive vectors for tumor targeting.
The following aspects are of importance in this regard.
(i) The specificity of the polyamine transport system
is not stringent. A wide variety of synthetic polyamine
derivatives and analogues are capable of using this
system.2
(ii) The activity of the polyamine transport system is
higher in transformed cells than in their normal coun-
terparts.3,4 In vivo, tissues with a high transport rate,
such as liver, and tissues with a high demand for
polyamines, such as tumors5 and prostate,6 take up
radiolabeled polyamines in greater amounts than other
tissues.
(iii) Depletion of intracellular polyamine concentra-
tions, e.g., by 2-(difluoromethyl)ornithine (DFMO), an
* To whom correspondence should be addressed. Phone: 33 (0) 2
99 33 62 33. Fax: 33 (0) 2 99 33 68 99. E-mail: delcros@univ-rennes1.fr.
† UPR ESA CNRS 6027.
‡ UMR CNRS 6510.
10.1021/jm010897q CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/18/2001

3654 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Martin et al.
Sch em e 1a
F igu r e 1. N-Benzylpolyamines.
as borocaptate (BSH) and p-borophenylalanine (BPA),
which are in clinical trial. However, the cytotoxicity of
the carboranes prohibits their use. Fluorine-containing
polyamines have been suggested for noninvasive imag-
ing of tumors by 18F positron emission tomography
(PET; for reviews, see refs 22 and 23) or by 19F NMR.
Various fluorinated putrescine derivatives have been
synthesized for this purpose.24-27 Finally, iodine-
substituted diamines were studied for application in 125I
scintigraphy.28,29
a
Reagents: (a) XPhCHO, 3 Å molecular sieves, Et2O; (b)
NaBH4, EtOH; (c) HCl (12 M), EtOH; (d) PhB(OH)2, Et2O/H2O.
protected 1,4-diaminobutane 1, obtained by reaction of
(Boc)2O with a large excess of 1,4-diaminobutane,34 was
employed as starting material. Reductive amination of
1 to 3 was achieved in two steps: imine 2 was obtained
from 1 and benzaldehyde derivatives, and was then
further reduced to 3 with NaBH4. 2-(4-Formylphenyl)-
5,5-dimethyl-1,3,2-dioxaborane was prepared from 4-bro-
mobenzaldehyde by a described procedure.35 The tert-
butoxycarbonylprotectinggroupwasremovedquantitatively
by acidic treatment of 3. Impurities from 3 were
completely removed by organic extractions, to afford
products 4 of high purity. Boronic acid 5 from 2,2-
dimethylpropane-1,3-diol boronate ester 4c was pre-
pared by reacting phenylboronic acid in a two-phase
ether/water system.36 This procedure permits 5 to be
obtained in high purity owing to the differences in
solubility. The starting 2,2-dimethylpropane-1,3-diol
ester 4c and the free boronic acid are soluble in the
aqueous phase while the 2,2-dimethylpropane-1,3-diol
phenylboronate ester accumulates in the organic phase.
N-{3-[(4-Aminobutyl)amino]propyl}(phenylmethyl)-
amine trihydrochloride (10a , N1-bz-sp d ), N-{3-[(4-ami-
nobutyl)amino]propyl}[(4-fluorophenyl)methyl]amine tri-
hydrochloride (10b, N1-4-F bz-sp d ), and N-{3-[(4-amino-
bu t yl)a m in o]pr opyl}{[4-(dih ydr oxybor yl)ph en yl]-
methyl}amine trihydrochloride (11, N1-4-Bbz-sp d ) were
synthesized by the route illustrated in Scheme 2 from
3-[(4-aminobutyl)amino]propanenitrile37 (6). tert-Butyl
N-(3-aminopropyl)-N-{4-{[(tert-butyloxy)carbonyl]amino}-
butyl}carbamate (7) was obtained from 6 according to
the literature38 by protection of the primary and second-
ary amino groups with a Boc, and reduction of the nitrile
group. Benzylation of 7 with substituted benzaldehydes
was effected by reductive amination in high yields.
Removal of the Boc groups by acidic cleavage afforded
the target compounds 10a and 10b. N1-4-Bbz-sp d (11)
was obtained from 10c using the same process as that
used for the preparation of 5.
Because conjugates of spermine exhibit high toxic-
ity,20,30 we focused our effort on putrescine and sper-
midine derivatives. To be taken up via the polyamine
transport system, spermidine derivatives should pref-
erentially be substituted on the N1-amino group, so that
the aminobutyl moiety is free to interact with the
transporter,31,32 but N4-substituted spermidines are also
substrates of the polyamine transport system.33 On the
basis of these results, we envisaged to prepare spermi-
dine derivatives carrying a heterosubstituted benzyl
substituent, either on the terminal nitrogen (N1-bz-sp d ,
N1-4-F bz-sp d , N1-4-Bbz-sp d ) or on the central nitrogen
(N4-bz-sp d , N4-4-Bbz-sp d , N4-3-Bbz-sp d ) (Figure 1) as
potential agents for BNCT or PET imaging. Boron- or
fluorine-substituted N-benzylputrescines (bz-pu t, 4-Fbz-
p u t, and 4-Bbz-p u t) were also synthesized. Boron and
fluorine were preferentially introduced in the para-
position of the aromatic moiety, because of the greater
stability of the resulting products. In vitro biological
properties of these compounds were evaluated in com-
parison with the unsubstituted parent N-ben-
zylpolyamines. This included DNA-binding, cytotoxicity,
and the ability to use the polyamine transport system.
The accumulation of the compounds in target cells is
also reported.
Ch em ica l Syn th eses
Syntheses of the target compounds N-(4-aminobutyl)-
(phenylmethyl)amine dihydrochloride (4a, bz-pu t), N-(4-
aminobutyl)[(4-fluorophenyl)methyl]amine dihydrochlo-
ride (4b, 4-F bz-p u t), and N-(4-aminobutyl){[4-
(dihydroxyboryl)phenyl]methyl}amine dihydrochloride
(5, 4-Bbz-p u t) are shown in Scheme 1. The mono-Boc-

N-Benzylpolyamines in Cancer Therapy and Imaging
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3655
Sch em e 2a
Sch em e 3a
a
Reagents: (a) Boc-ON, THF; (b) 2,2-dimethylpropane-1,3-diol,
Et2O; (c) NBS, AIBN, CCl4; (d) 13a or 13b or PhCH2Br, NaHCO3,
NaI; (e) HCl (12 M), EtOH.
a
Ta ble 1. DNA Binding Affinity of N-Benzylpolyamine
Reagents: (a) (Boc)2O, THF; (b) H2, 5 bar, Raney-Ni, EtOH,
Derivativesa
NaOH; (c) XPhCHO, 3 Å molecular sieves, Et2O; (d) NaBH4, EtOH;
(e) HCl (12 M), EtOH; (f) PhB(OH)2, Et2O/H2O.
compound
IC50 (µM)b
compound
IC50 (µM)b
putrescine
bz-p u t
4-F bz-p u t
4-Bbz-p u t
spermidine
>11000
N1-bz-sp d
107 ( 9
341 ( 42
37 ( 2
The desired target compounds N-(3-aminobutyl)-N(3-
aminopropyl){{4-(5,5-dimethyl[1,3,2]dioxaborinan-2-yl)-
phenyl}methyl}amine trihydrochloride (15a , N4-4-Bbz-
sp d ), and N-(3-aminobutyl)-N-(3-aminopropyl){{3-(5,5-
dim et h yl[1,3,2]dioxa bor in a n -2-yl)ph en yl}m et h yl}-
amine trihydrochloride (15b , N4-3-Bb z-sp d ) were
obtained according to a convergent synthetic route
illustrated in Scheme 3 from spermidine and phenyl
boronic acid derivatives. The tert-butyl N-{4-{N-{3-[(tert-
butyloxycarbonyl)amino]propyl}amino}butyl}carba-
mate (12) was first synthesized from the free triamine
using 2-[(tert-butoxycarbonyl)oxyimine]-2-phenylaceto-
nitrile (Boc-ON), to selectively protect the primary
amino groups.32 The 2,2-dimethylpropane-1,3-diol ester
of the [4-(bromomethyl)phenyl]- and [3-(bromomethyl)-
phenyl]boronic acids 13a and 13b were prepared by
halogenation of the corresponding (4-methylphenyl)-
and (3-methylphenyl)boronate with N-bromosuccinim-
ide.39 Alkylation of 12 was performed in the presence
of NaHCO3 and NaI. Deprotection of the resulting
spermidine derivatives 14 was achieved with aqueous
concentrated hydrochloric acid in ethanol. N-(3-Ami-
nobutyl)-N-(3-aminopropyl)(phenylmethyl)amine40 tri-
hydrochloride (15c, N4-bz-sp d ) was prepared in a
similar way from 12 and benzyl bromide. It should be
noted that the N-benzylated spermidines 15 are quite
hygroscopic.
3109 ( 765
>3000
N1-4-F bz-sp d
N1-4-Bbz-sp d
N4-bz-sp d
1382 ( 243
98 ( 20
310 ( 39
102 ( 0.6
N4-4-Bbz-sp d
a
As measured by displacement of ethidium bromide from calf
b
thymus DNA. Concentration of N-benzylpolyamine derivative
required to decrease fluorescence intensity of the DNA-ethidium
bromide complex by 50%. (Data are expressed as mean ( SD).
Biologica l Stu d ies
1. DNA Bin d in g Assa y. IC50 values, defined as the
concentration of a polyamine derivative required to
decrease the fluorescence of the ethidium bromide-
DNA complex by 50%, are reported in Table 1. As
observed with their natural counterparts, spermidine
derivatives were better ligands for DNA than the
corresponding putrescine derivatives. Notably, N1-bz-
sp d exhibited an affinity for DNA equivalent to that of
spermidine, whereas N4-bz-sp d was 3 times less ef-
ficient. Addition of a boron group to the parent N-
benzylpolyamines increased the ability to displace ethid-
ium bromide. N1-4-Bbz-sp d exhibited about a 2.5 times
higher affinity for DNA than the natural spermidine.
In contrast, fluorinated compounds displayed a weaker
affinity for DNA than the parent compounds.
2. Ability of P olya m in e Der iva tives To Use th e
P olya m in e Tr a n sp or t System . The ability of the
derivatives to be taken up by active transport was

3656 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Martin et al.
Ta ble 2. Accumulation of N-Benzylpolyamine Derivatives in
Ta ble 4. Polyamine Levels in 3LL Cells after Treatment with
N-Benzylpolyamine Derivativesa
CHO and Polyamine Transport Deficient CHO-MG Cellsa
dose CHO (nmol/mg CHO-MG (nmol/ ratio CHO/
putrescine spermidine
spermine
(nmol/mg
of protein)
(µM)
of protein)
mg of protein)
CHO-MG
dose (nmol/mg
(nmol/mg
drug
(µM) of protein) of protein)
bz-p u t
50
50
50
50
10
0.40 ( 0.03 0.10 ( 0.02b
0.90 ( 0.05 0.45 ( 0.04b
5.76 ( 0.25 0.51 ( 0.06b
4.0
2.0
controlb none
0.74 ( 0.21 9.28 ( 1.17
7.24 ( 0.55
4-F bz-p u t
4-Bbz-p u t
N1-bz-sp d
N1-4-F bz-sp d
N1-4-Bbz-sp d
N4-bz-sp d
N4-4-Bbz-sp d 50 0.12 ( 0.03 15.54 ( 0.73 11.28 ( 0.39
11.3
>35
>19
>9.6
2.5
DFMOc none
bz-p u t
Bdl
50 Bdl
50 Bdl
50 Bdl
bdl
0.89 ( 0.07
0.92 ( 0.27 10.99 ( 2.30
1.07 ( 0.08
6.68 ( 0.67
5.79 ( 0.11
17.58 ( 2.8
bdlb
9.40 ( 0.89 bdlb
4.78 ( 0.46 bdlb
4-F bz-p u t
4-Bbz-p u t
50
250
13.64 ( 2.60 5.55 ( 0.72b
5.05 ( 0.68 3.84 ( 0.87b
5.65 ( 0.02 3.71 ( 0.09b
7.52 ( 0.002
7.66 ( 0.20
7.23 ( 0.29
8.25 ( 1.11
7.25 ( 0.31
8.64 ( 0.60
7.40 ( 0.07
N4-4-Bbz-sp d 250
N4-3-Bbz-sp d 250
1.3
1.5
250 1.31 ( 0.30 2.28 ( 0.00
N1-bz-sp d
10 Bdl
Bdl
bdl
bdl
0.14 ( 0.07
4.15 ( 0.46
8.92 ( 0.88
N1-4-F bz-sp d
1
a
N1-4-Bbz-sp d 50 Bdl
CHO and CHO-MG cells were incubated for 24 h with the
compounds. Levels of N-benzylpolyamine derivatives were deter-
mined by HPLC (data are expressed as mean ( SD). bdl ) below
N4-bz-sp d
N4-4-Bbz-sp d 50 Bdl
250 Bdl
b
detection limit (<0.5 nmol/mg of protein). p < 0.01: significantly
a
The polyamine content was evaluated by HPLC (data are
different from values for CHO cells.
b
expressed as mean ( SD). bdl ) below detection limit. 3LL cells
were incubated for 24 h in the presence or absence of N4-4-Bbz-
sp d before processing. c 3LL cells were incubated for 24 h with
polyamine derivatives after a 24 h preincubation period in the
presence of 5 mM DFMO.
Ta ble 3. Effect of N-benzylpolyamine Derivatives, BSH, and
BPA on 3LL Cell Growth
effect of DFMO
IC50 (µM)a,e
pretreatmentb,e
72 h
48 h
48 h
did not show any cytotoxicity on 3LL cells up to 1 mM.
N-Benzylputrescine exhibited a very moderate effect on
3LL cell growth (IC50 ≈ 600 µM). Its fluorinated
analogue 4-F bz-p u t was slightly more cytotoxic, while
the boron-containing derivative did not affect cell growth
up to 1 mM. The boronated compounds appear even less
cytotoxic than BSH.
Most of the benzyl derivatives had no or a limited
effect on intracellular polyamine pools. At cytotoxic
concentrations, N1-benzylspermidine and its fluorine
analogue induced a significant decrease of intracellular
putrescine levels but did not affect spermidine and
spermine concentrations (data not shown). Both N4-4-
Bbz-sp d and N4-3-Bbz-sp d derivatives induced a de-
crease in putrescine and an approximate increase of
both spermidine and spermine concentrations by 60%
(Table 4), whereas N4-bz-sp d did not affect polyamine
levels at the same concentration.
The influence of DFMO on the cytotoxicity of the
polyamine derivatives was studied by monitoring 3LL
cell growth after a 24 h preincubation with 5 mM
DFMO, followed by a 48 h exposure to the drugs (Table
3). Combined exposure with DFMO greatly enhanced
the cytotoxicity of N1-benzylspermidine and of its flu-
orinated analogue. In the presence of DFMO, the IC50
values of N1-bz-sp d and N1-4-F bz-sp d were diminished
by 90%. In contrast, all the other N-benzyl derivatives
antagonized DFMO-induced cell growth arrest; i.e., they
supported growth of putrescine- and spermidine-deplet-
ed cells, as shown in Figure 2 for spermidine and N4-
benzylspermidine derivatives. However, total reversion
of the DFMO effect required 10 and 100 times higher
concentrations of N4-4-Bbz-sp d and N4-bz-sp d , respec-
tively, than of spermidine. The putrescine derivatives
as well as N1-4-Bbz-sp d reversed only partially the
effect of DFMO (see Figure 2).
In 3LL cells pretreated with DFMO, intracellular
putrescine and spermidine were below the detection
level (Table 4). Polyamine pools remained unchanged
in 3LL cells treated with N1-bz-sp d and N1-4-F bz-sp d ,
the derivatives showing a synergistic effect with DFMO.
In contrast, significant amounts of spermidine were
detected in cells treated with the other derivatives. In
cells treated with 50 µM N4-4-Bbz-sp d , spermidine
incubation incubation
incubation
BSH
147 ( 50 287 ( 20 NDc
>100
BPAd
>100
NDc
bz-p u t
604 ( 32 993 ( 62 partial reversion (>50 µM)
468 ( 21 568 ( 64 partial reversion (>10 µM)
4-F bz-p u t
4-Bbz-p u t
N1-bz-sp d
>1000
>1000
partial reversion (>50 µM)
9.3 ( 0.9 31.0 ( 1.0 synergy (IC50 ) 11.9 ( 3.6 µM)
N1-4-F bz-sp d 2.3 ( 0.8 4.7 ( 1.1 synergy (IC50 ) 0.5 ( 0.3 µM)
N1-4-Bbz-sp d 400 ( 35 824 ( 176 partial reversion (>1 µM)
N4-bz-sp d
>1000
>1000
>1000
>1000
complete reversion (>100 µM)
complete reversion (>10 µM)
NDc
N4-4-Bbz-sp d >1000
N4-3-Bbz-sp d >1000
a
Compounds were added to the culture medium 24 h after
seeding. Twenty-four hours after seeding, 5 mM DFMO was
b
added for a 24 h preincubation period before addition of other
drugs. c ND ) not determined. Because of its poor solubility, the
d
cytotoxicity of BPA was not tested at concentrations superior to
100 µM. e All data are expressed as mean ( SEM.
determined by comparing their accumulation in CHO
cells and in mutant polyamine transport deficient CHO-
MG cells. As shown in Table 2, at concentrations that
did not affect cell viability, all derivatives were more
actively taken up by CHO than by CHO-MG cells. The
highest CHO/CHO-MG accumulation ratios were ob-
served with the N1-benzylspermidine derivatives. These
were not detected in CHO-MG cells. In contrast, the
accumulation ratios for the N4-spermidine derivatives
were low, despite their considerable accumulation in
CHO cells, which was of the same order of magnitude
as that of the N1-derivatives. Among the putrescine
derivatives, 4-Bbz-p u t showed the greatest accumula-
tion in CHO cells and an accumulation ratio superior
to 11.
3. Stu d ies on Lew is Lu n g Ca r cin om a Cells (3LL
Cells). Effect of P olya m in e Der iva tives on Cell
Gr ow th a n d P olya m in e Meta bolism of 3LL Cells.
The effect of the drugs on the growth of 3LL cells was
evaluated using an MTT assay. The concentrations of
the drugs causing 50% cell growth inhibition (IC50) are
reported in Table 3.
Among the N1-benzylspermidines, the fluorine-con-
taining derivative was the most cytotoxic (IC50 around
2 µM at 72 h), followed by the compound without a
substituent on the benzyl moiety, while the boronated
derivative showed considerably less growth inhibition
(IC50 around 400 µM at 72 h). The N4-benzylspermidines

N-Benzylpolyamines in Cancer Therapy and Imaging
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3657
F igu r e 2. Reversion of the cytostatic effect of DFMO by spermidine and N-benzylspermidines in 3LL cells: (A) effect of spermidine
and N4-bz-sp d ; (B) effect of N4-4-Bbz-sp d and N1-4-Bbz-sp d . Twenty-four hours after seeding, 5 mM DFMO was added to the
3LL cell culture medium for 24 h, followed by a 48 h incubation period with spermidine or a spermidine derivative. The percent
cell growth was calculated from the value of the cell growth of control cells treated only with DFMO.
Ta ble 5. Effect of a DFMO Pretreatment on the Accumulation of N-benzyl Polyamine Derivatives in 3LL Cells
HPLC measurementsc
ICP-AES measurementsc
boron (nmol/mg of protein)
drug (nmol/mg of protein)
ratio
with/without
DFMO
dose
(µM)
without
DFMOa
with
without
DFMOa
with
drug
DFMOb
DFMOb
BSH
BPA
bz-p u t
4-F bz-p u t
4-Bbz-p u t
50
50
50
50
10
50
10
1
10
50
10
50
250
50
250
250
ND
ND
ND
ND
ND
ND
3.0
2.4
6.5
6.4
2.7
2.2
ND
2.1
2.1
1.4
0.9
0.4
0.8
0.9
0.82 ( 0.30
0.80 ( 0.09
ND
ND
0.34 ( 0.19
0.55 ( 0.06
0.52 ( 0.01
1.32 ( 0.27
9.00 ( 0.46
5.51 ( 0.29
12.87 ( 1.18
2.01 ( 0.73
2.59 ( 0.04
4.92 ( 0.00
6.11 ( 0.41
2.60 ( 0.50
3.35 ( 0.51
4.52 ( 0.67
1.03 ( 0.22d
1.31 ( 0.04d
3.37 ( 0.06d
8.44 ( 1.27d
23.67 ( 2.46d
12.19 ( 2.37d
ND
1.29 ( 0.10
2.70 ( 1.22
3.46 ( 1.13
6.73 ( 1.69
5.68 ( 0.43
1.83 ( 0.20e
N1-bz-sp d
N1-4-F bz-sp d
N1-4-Bbz-sp d
N4-bz-sp d
4.18 ( 1.27d
5.52 ( 0.36d
6.92 ( 0.66d
5.46 ( 0.35
0.98 ( 0.17
2.55 ( 0.05
3.93 ( 0.26
N4-4-Bbz-sp d
N4-3-Bbz-sp d
a
b
Cells were incubated for 24 h with the derivatives or BSH. After a 24 h incubation period with 5 mM DFMO, cells were further
incubated for 24 h with the N-benzylpolyamine derivatives. c ND ) not done. Data are expressed as mean ( SD. p < 0.01: significantly
d
different from control values without DFMO. e p < 0.01: significantly different from values measured by HPLC.
levels were restored to normal values, but putrescine
remained undetectable. In contrast, in cells treated with
250 µM 4-Bbz-p u t, putrescine pools were replenished.
Accu m u la tion of P olya m in e Der iva tives in 3LL
Cells. Intracellular levels of the polyamine derivatives
were determined in 3LL cells after a 24 h exposure at
concentrations that did not affect cell growth, by HPLC
of the respective compound. The accumulation of the
derivatives in cells pretreated with DFMO for 24 h is
also reported (Table 5).
from 10 to 250 µM. For N4-4-Bbz-sp d , the ratio re-
mained below 1 in the range 50-250 µM.
Measurement of the cellular boron content was per-
formed in 3LL cells treated with the boron-containing
analogues (Table 5). For all boron-containing polyamines,
the boron content was similar to that of the amount of
the drug determined by HPLC. The same observation
was made in DFMO-treated cells, except in cells exposed
to N4-4-Bbz-sp d , where the boron content was signifi-
cantly higher. All boron-containing polyamine deriva-
tives allowed boron accumulations similar to or higher
than those of BSH and BPA. When 3LL cells were
pretreated with DFMO, boron concentrations with
polyamine derivatives were 2-8-fold higher than with
BSH or BPA.
At identical extracellular concentrations, 3LL cells
accumulated higher amounts of spermidine than of the
putrescine derivatives. The fluorine-containing polyamine
derivatives exhibited accumulation in 3LL cells compa-
rable to that of their parent compounds. In contrast, the
boron-containing moiety enhanced the accumulation of
the putrescine derivative but diminished that of the
spermidine derivatives.
4. Accu m u la tion of Bor on a ted P olya m in es in
Va r iou s Cell Lin es. As melanoma and brain tumors
are the main targets for BNCT, accumulation of boron-
containing derivatives was studied in murine C6 glio-
blastoma and B16 melanoma cells, as well as in human
U251 glioblastoma cells (Table 6). 4-Bbz-p u t and N1-
4-Bbz-sp d accumulated more efficiently in B16 cells
than in C6 and U251 cells. In contrast, accumulation
of N4-4-Bbz-sp d was superior in glioblastoma cells.
Pretreatment of the cells with DFMO enhanced the
Pretreatment of 3LL cells with DFMO enhanced the
accumulation of all derivatives except that of N4-4-Bbz-
sp d . The highest difference between DFMO-treated and
control cells was observed with 4-Bbz-p u t (ratio around
6, while it was only around 2 for the other derivatives).
With N4-bz-sp d , the accumulation ratio decreased from
2.1 to 0.9 when concentrations in the medium increased

3658 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Martin et al.
Ta ble 6. Accumulation of Boron-Containing N-Benzylpolyamine Derivatives in Melanomaa and Glioblastomab Cells
drug (50 µM,
24 h exposure)
C6
U251
B16
(nmol/mg of protein)c
(nmol/mg of protein)c
(nmol/mg of protein)c
4-Bbz-p u t
controld
0.09 ( 0.02
0.41 ( 0.03f
4.6
1.63 ( 0.03
3.51 ( 0.06f
2.2
2.92 ( 0.22
2.50 ( 0.25
0.9
0.21 ( 0.07
0.22 ( 0.01
1.1
1.65 ( 0.04
2.52 ( 0.08f
1.5
2.12 ( 0.03
1.79 ( 0.02f
0.8
5.77 ( 0.47
20.41 ( 0.05f
3.5
4.75 ( 0.11
9.26 ( 0.95f
2.0
0.92 ( 0.24
0.45 ( 0.03
0.5
DFMOe
ratio ( DFMO
N1-4-Bbz-sp d
N4-4-Bbz-sp d
controld
DFMOe
ratio ( DFMO
controld
DFMOe
ratio ( DFMO
a
b
Murine B16 melanoma cells. Rat C6 glioblastoma cells and human U251 glioblastoma cells. c Levels of N-benzylpolyamine derivatives
d
were measured by HPLC (data are expressed as mean ( SD). Cells were incubated with 50 µM boron-containing polyamine for 24 h.
e After a 24 h incubation period with 5 mM DFMO, cells were further incubated for 24 h with 50 µM polyamine derivatives. f p < 0.01:
significantly different from control values without DFMO.
accumulation of 4-Bbz-p u t in C6 and B16 cells about
4 times, but had no effect in U251 cells. DFMO pre-
treatment increased the accumulation of N1-4-Bbz-sp d
by a factor of 2 in all three cell lines. In contrast, N4-
4-Bbz-sp d amounts were in all cell lines similar or
lower in DFMO-treated vs normal cells.
another transport system, or more likely that plasma
membranes are more permeable to this compound.
Fluorine substitution on the benzyl moiety of both bz-
p u t and N1-bz-sp d did not alter greatly cellular uptake.
In contrast, the boronic acid substitution, a bulkier
substituent, had a significant impact on uptake. It
reduced 2-3 times the accumulation of the spermidine
derivatives in CHO and 3LL cells. The boronate group
mainly affects the ability of the drug to use the
polyamine transporter since polyamine transport defi-
cient cells accumulated similar amounts of N4-4-Bbz-
sp d and N4-bz-sp d . The position of the substituent on
the benzyl moiety has little influence on transport as is
evident from the similar behavior of N4-4-Bbz-sp d and
N4-3-Bbz-sp d . In contrast, the boronic substitution of
bz-p u t considerably increased its cellular uptake.
Another argument in favor of the use of the polyamine
transport system by the polyamine derivatives is their
increased accumulation in polyamine-depleted cells.
Polyamine depletion results in the up-regulation of the
polyamine transport system. Depletion of putrescine
and spermidine by treatment with DFMO is known to
increase the accumulation rate of polyamine-related
drugs.33,46-48 The uptake of all polyamine derivatives
reported here was enhanced in DFMO-treated 3LL cells,
except for N4-spermidine derivatives. This discrepancy
will be discussed later.
Among the three parent compounds, only N1-bz-sp d
exhibited a significant cytotoxicity on 3LL cells, with
an IC50 around 10 µM. This is in agreement with
previous studies showing that N1-substituted sper-
midines and spermine were more toxic than their N4-
substituted homologues.20 As has been previously men-
tioned, introduction of fluorine into the benzene moiety
increased and a boronic acid substituent decreased the
cytotoxicity of the derivative. This effect was most
striking in the N1-spermidine series. The reduced toxic-
ity is presumably related to the lower lipophilicity of
the derivative as evidenced by a significant reduction
in the chromatographic retention time on a reversed-
phase column as ion pairs with n-octanesulfonic acid
(N1-4-Bbz-sp d , 22.9 min; N1-bz-sp d , 28.1 min; N1-4-
F bz-sp d , 28.4 min; 4-Bbz-p u t, 17.0 min; bz-p u t, 23.2
min; 4-F bz-p u t, 24.88 min). Reduced lipophilicity may
impair interactions with intracellular sites involved in
growth support.
Discu ssion
The rationale behind the synthesis of new N-ben-
zylpolyamines was to develop compounds sharing some
properties of the natural polyamines, particularly their
low cytotoxicity and their ability to be taken up by cells
via a specific polyamine transport system. The up-
regulation of the activity of this transport system in
cancer cells after treatment with DFMO allows the
preferential accumulation of drugs in the tumor cells if
they are transported by this system. High tumor/normal
tissue ratios are a requirement for drugs used in BNCT
as well as for probes used in imaging.
All three parent N-benzylpolyamines use the poly-
amine transport system as became obvious from the
greater accumulation in CHO cells than in CHO-MG
mutant cells, which lack the polyamine transport sys-
tem. However, uptake was highly dependent on the
nature of the polyamine and the position of the benzyl
moiety on the polyamine chain. Earlier studies demon-
strated a relationship between the number of protonated
nitrogens of the polyamine derivative and its affinity
for the polyamine transport system.41-45 The higher
number of positive charges is the reason for the more
efficient accumulation of the triamines N1-bz-sp d and
N4-bz-sp d in comparison with that of the diamine bz-
p u t . Despite comparable accumulation of N1-b z-sp d
and N4-bz-sp d in CHO cells, their accumulation in the
mutant CHO-MG cells was strikingly different. N1-bz-
sp d levels were below the detection limit. Its entry into
cells appears to be entirely dependent on the presence
of an active polyamine transport system. In contrast,
CHO-MG cells accumulated significant amounts of N4-
bz-sp d ; the relative accumulation ratio between CHO
and CHO-MG cells did not exceed 4 (while it was
superior to 35 with N1-bz-sp d ). A greater importance
of the polyamine transport system in the uptake of the
N1-derivative is in agreement with previous studies,
which showed a higher affinity for the transporter of
N1-spermidine derivatives when compared to their N4-
homologues.31,32,43 However, the ability of N4-bz-sp d to
accumulate independently of the presence of an active
polyamine transporter indicates either the presence of
Combined exposure of cells to DFMO and to one of
the two cytotoxic drugs N1-bz-sp d and N1-4-F bz-sp d
produced a synergistic cytotoxic effect, most probably

N-Benzylpolyamines in Cancer Therapy and Imaging
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3659
due to the higher amounts of drug accumulating in
DFMO-treated cells. In contrast, all other drugs re-
versed partially or completely the cytostatic effect of
DFMO. In those cells, putrescine and/or spermidine
could be detected, while they were below the detection
limit in DFMO-treated cells not exposed to the drugs.
Incubation of the drugs in culture medium at 37 °C for
several days did not lead to the formation of free
polyamines. This indicates that the putrescine and
spermidine detected in DFMO-treated cells were formed
inside the cells. The newly formed putrescine and
spermidine counteract growth inhibition by DFMO. It
is known that N-benzyl derivatives of putrescine49 and
N,N′-bis(benzyl)polyamine analogues50 are substrates
of polyamine oxidase, which splits the conjugates into
the parent amine and benzaldehyde. It is likely that
both fluorinated and boronated putrescine and N1-
spermidine derivatives are also substrates of this oxi-
dase. In support of this assertion, we observed that, in
CHO cells, intracellular levels of bz-p u t or 4-Bbz-p u t
were 2-3-fold higher in the presence of MDL 72527, an
inhibitor of polyamine oxidase51 (data not shown).
In contrast to a previous report,33 N4-bz-sp d was
found to reverse the effect of DFMO. However, the
reversion was observed only in cells treated with at least
100 µM drug, a dose higher than the one used in the
previous study. A similar effect was observed with its
boron-containing derivative at 10 times lower concen-
tration. In cells treated with DFMO and with the two
derivatives, repletion of the spermidine pool was ob-
served. In DFMO-treated cells, 50 µM N4-4-Bbz-sp d
produced spermidine levels equivalent to those mea-
sured in normal cells. In addition, the boron content of
the cells was superior to the amount of drug, as
determined by HPLC. These observations strongly sug-
gest that free spermidine is formed as the result of
intracellular catabolism of N4-4-Bbz-sp d . Its degrada-
tion also occurs in normal cells, as is evident from the
increased spermidine and spermine levels after expo-
sure of cells to N4-4-Bbz-sp d . The enhanced formation
of spermidine in cells treated with N4-4-Bbz-sp d indi-
cates that the presence of the boron moiety on the benzyl
group of N4-bz-sp d favors its degradation. In cells
treated with the N1-benzylspermidine derivatives, in-
tracellular spermidine formation was observed only in
the case of the boron-containing derivatives. The cata-
bolic pathway responsible for this degradation remains
to be clarified. The repletion of spermidine pools ob-
served in cells treated with the N4-spermidine deriva-
tives is likely to be responsible for the prevention of the
up-regulation of the polyamine transport activity by
DFMO. This, along with the intracellular degradation
of the drugs, explains why DFMO did not improve the
accumulation of the N4-benzylspermidine derivatives.
pounds, the N1-4-F bz-sp d derivative is probably a good
candidate for PET because of its high accumulation in
cancer cells via the polyamine transport system. Its
cytotoxicity may not prevent its use, since in PET the
concentrations to be administrated are low.
P ot en t ia ls of t h e Bor on -Con t a in in g Ben zyl-
p olya m in es in BNCT. To become a candidate for
BNCT, a boron-containing drug should be nontoxic,
allow production of an intracellular boron content >10
µg/g in cancer tissue, and exhibit high tumor selectivity.
As reported above, none of our boron-containing drugs
show significant toxicity to 3LL cells. They were con-
siderably less cytotoxic than the clinically used BSH
(IC50 ) 150 µM, Table 2), and they appear to be less
toxic than the previously reported carborane-containing
polyamines (IC50 to F98 cells below 100 µM after 24 h
of treatment20,21). The three boron-containing ben-
zylpolyamines allowed us to accumulate boron to around
2 µg/g of 3LL cells. Despite the fact that each molecule
carries only 1 boron atom, the amount of boron ac-
cumulated was higher than that achieved with BSH
(only 0.7 µg of B/g of 3LL cells), a molecule carrying 12
boron atoms, and with BPA (0.9 µg of B/g of 3LL cells).
Because the uptake of 4-Bbz-p u t and N1-4-Bbz-sp d is
highly dependent on the polyamine transport activity,
pretreatment of 3LL cells with DFMO improved boron
accumulation (up to 6 µg of B/g of cells). The high
affinity of the polyamines to DNA14 is another property
of interest in BNCT. N1-4-Bbz-sp d exhibited higher
affinity for DNA than spermidine. This property allows
vectorization of boron into the nuclei and thus should
favor its efficacy in BNCT, as cell killing is thought to
be more effective with boron close to DNA.53
Targets for BNCT include brain tumors and mela-
noma. Murine as well as human glioblastoma cells
accumulated only low levels of 4-Bbz-p u t, N1-4-Bbz-
sp d , and N4-4-Bbz-sp d even after pretreatment with
DFMO. Only up to 3 µg of B/g of cells accumulated
within 24 h when treated with 0.5 µg of B/mL of
medium. Previously, the in vitro and in vivo accumula-
tion of the acid form of N4-4-Bbz-sp d in glioma cells
was reported.21 F98 cells accumulated around 100 µg
of B/g of cells after 48 h of incubation with 5.1 µg of
B/mL of medium. However, in mice bearing intracere-
bral GL261 gliomas, this compound (delivered by con-
tinuous subcutaneous infusion by an Alzet pump) does
not allow sufficient boron accumulation in the tumor
tissue. Other N4-spermidine derivatives carrying a
carborane moiety (10 boron atoms) did not produce
higher boron concentrations in F98 glioma cells in vitro.
In vivo, the boron content in the GL261 tumor was only
7 µg of B/g of tumor after infusion with the carborane
analogue of N4-4-Bbz-sp d .21 Up to now, in vivo assays
with boron-containing N1-spermidine or N-putrescine
derivatives have not been reported. However, our results
suggest that boron-containing benzylpolyamines may
not be suitable for BNCT of brain tumors.
P ot en t ia ls of t h e F lu or in e-Con t a in in g Ben -
zylp olya m in es in P ET. As 18F is a short-lived isotope
(half-life 110 min), preparation of 18F-labeled compounds
useful for PET should not exceed 2 h. Syntheses of
4-F bz-p u t and N1-4-F bz-sp d are based on reductive
amination of selectively protected polyamines with
4-fluorobenzaldehyde. This efficient, regioselective, and
rapid sequence might be applicable to the corresponding
labeled compound, since the [18F]benzaldehyde can
easily be prepared.52 Among the two fluorinated com-
In B16 melanoma cells, the polyamine derivatives
4-Bbz-p u t and N1-4-Bbz-sp d allowed the accumulation
of boron up to 5 µg of B/g of cells with only 0.55 µg of
B/mL in the medium. As expected from their ability to
use the polyamine transport system, pretreatment with
DFMO increased boron accumulation in B16 cells (up
to 18 µg of B/g of B16 cells). In a previous report, B16

3660 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Martin et al.
Da ta for ter t-Bu tyl N-{4-[(P h en ylm eth ylid en e)a m in o]-
bu tyl}ca r ba m a te (2a ): prepared from 1 and benzaldehyde;
colorless oil; 98%; H NMR (200 MHz, CDCl3) δ 8.28 (s, 1H),
cells exposed for 18 h to BPA or BSH at 5 or 10 µg of
boron/mL of medium were shown to accumulate 12.5
µg of B/g of cells (results estimated on the basis of 2
mg/106 B16 cells).54 Others have reported boron con-
centrations of 20 µg/g of B16 cells after treatment with
BPA at 11 µg of B/mL of medium.55 These results
suggest 4-Bbz-p u t and N1-4-Bbz-sp d for BNCT of
melanomas.
1
7.80-7.65 (m, 2H), 7.45-7.30 (m, 3H), 4.85 (br, 1H), 3.61 (t, J
) 6.7 Hz, 2H), 3.17 (t, J ) 6.2 Hz, 2H), 1.80-1.50 (m, 4H),
1.44 (s, 9H); 13C NMR (50 MHz, CDCl3) δ 161.1, 156.0, 136.1,
130.6, 128.6, 128.0, 78.9, 61.1, 40.3, 28.4, 28.1, 27.8.
Data for ter t-Bu tyl N-{4-{[(4-Flu or oph en yl)m eth yliden e]-
a m in o}bu tyl}ca r ba m a te (2b): prepared from 1 and 4-fluo-
robenzaldehyde; colorless oil; 95%; 1H NMR (200 MHz, CDCl3)
δ 8.17 (s, 1H), 7.80-7.58 (m, 2H), 7.08-6.92 (m, 2H), 4.65 (br,
1H), 3.60-3.50 (m, 2H), 3.18-3.00 (m, 2H), 1.75-1.30 (m,
13H); 13C NMR (50 MHz, CDCl3) δ 160.1, 155.0, 130.4, 130.2,
116.3, 115.8, 61.4, 39.3, 27.4, 27.1, 26.8; 19F NMR (282 MHz,
CDCl3) δ -110.160.
Da ta for ter t-Bu tyl N-{4-{{[4-(5,5-Dim eth yl[1,3,2]d ioxa -
bor in an-2-yl)ph enyl]m eth ylidene}am in o}bu tyl}car bam ate
(2c): prepared from 1 and 2-(4-formylphenyl)-5,5-dimethyl-
1,3,2-dioxaborane; colorless solid; 96%; mp ) 118-120 °C; 1H
NMR (200 MHz, CDCl3) δ 8.28 (s, 1H), 7.85 (d, J ) 8.1 Hz,
2H), 7.70 (d, J ) 8.1 Hz, 2H), 4.70 (br, 1H), 3.78 (s, 4H), 3.70-
3.58 (m, 2H), 3.25-3.10 (m, 2H), 1.82-1.40 (m, 13H), 1.05 (s,
6H); 13C NMR (50 MHz, CDCl3) δ 161.5, 156.0, 137.9, 134.1,
133.9, 127.2, 72.3, 61.3, 40.2, 28.4, 28.1, 27.9, 21.9; HRMS (EI)
calcd for C21H33BN2O4 (M+) 388.2533, found 388.2548.
Data for ter t-Bu tyl N-{3-{[(P h en yl)m eth yliden e]am in o}-
p r op yl}-N-{4-{[(ter t-b u t yloxy)ca r b on yl]a m in o}b u t yl}-
ca r ba m a te (8a ): prepared from 7 and benzaldehyde; colorless
oil; 95%; 1H NMR (200 MHz, CDCl3) δ 8.20 (s, 1H); 7.72-7.58
(m, 2H), 7.45-7.26 (m, 3H); 4.65 (br, 1H), 3.65-3.47 (m, 2H),
3.30-3.00 (m, 6H), 2.00-1.25 (m, 24H); 13C NMR (50 MHz,
CDCl3) δ 161.7, 156.4, 156.0, 136.6, 131.0, 128.9, 128.4, 79.6,
79.4, 59.5, 47.1, 45.6, 40.6, 28.8, 27.8, 26.1.
An essential requirement for drugs used in BNCT is
a high tumor selectivity in vivo. The ability of the drugs
to use the polyamine transport system is a basis for
tumor selectivity, in particular, in combination with
DFMO, which up-regulates the transport rate selec-
tively in the tumor tissue.11-13 As compared with N1-
spermidine derivatives, results reported in this study
have shown that the intracellular uptake of N4-spermi-
dine derivatives was poorly dependent on the polyamine
transport system. Previous biodistribution studies with
a N4-carborane-containing spermidine derivative re-
vealed that this compound allowed the accumulation of
boron concentrations in B16 melanoma in vivo to up to
18 µg of B/g of tumor.21 Tumor/blood and tumor/skin
ratios were superior to 3, but were not judged sufficient
for a clinical use by the authors. The most promising
derivatives described in this work are likely to be 4-Bbz-
p u t and N1-4-Bbz-sp d since their accumulation is
highly dependent on the presence of active transport,
especially since tumor selectivity can be improved by
pretreatment with DFMO. In addition, their solubility
in water and their low toxicity are advantages over the
currently used drugs.
Data for ter t-Bu tyl N-{3-{[(4-Flu or oph en yl)m eth yliden e]-
a m in o}p r op yl}-N-{4-{[(ter t-b u t yloxy)ca r b on yl]a m in o}-
bu tyl}ca r ba m a te (8b): prepared from 7 and 4-fluorobenzal-
The potentials of N-benzylpolyamines as vectors of
boron for tumor targeting are evidenced by the present
in vitro study. In vivo experiments are in progress on
murine melanoma tumors, to further evaluate N-ben-
zylpolyamines as potential drugs for BNCT. In addition,
the synthesis of new benzylpolyamine derivatives with
more than one boron atom is being designed.
1
dehyde; colorless oil; 96%; H NMR (200 MHz, CDCl3) δ 8.17
(s, 1H), 7.70-7.56 (m, 2H), 7.10-6.92 (m, 2H), 4.62 (br, 1H),
3.62-3.45 (m, 2H), 3.33-2.95 (m, 6H), 1.95-1.72 (m, 2H),
1.60-1.25 (m, 22H); 13C NMR (50 MHz, CDCl3) δ 160.2, 156.4,
156.0, 136.9, 130.4, 130.2, 116.2, 115.8, 79.6, 79.4, 59.4, 47.1,
45.6, 40.6, 28.8, 28.7, 27.8, 26.1; 19F NMR (282 MHz, CDCl3)
δ -110.250.
Da ta for ter t-Bu tyl N-{3-{{[4-(5,5-Dim eth yl[1,3,2]d ioxa -
bor in a n -2-yl)p h en yl]m eth ylid en e}a m in o}p r op yl}-N-{4-
{[(ter t-bu tyloxy)ca r bon yl]a m in o}bu tyl}ca r ba m a te (8c):
prepared from 7 and 2-(4-formylphenyl)-5,5-dimethyl-1,3,2-
Exp er im en ta l Section
Reagents were purchased from chemical companies and
used directly without further purification unless otherwise
specified. Diethyl ether (Et2O) and tetrahydrofuran (THF)
were distilled from deep blue solutions of sodium/benzophe-
none ketyl prior to use. All melting points were determined
on a Kofler apparatus and are uncorrected. NMR spectra were
recorded on Bruker AC 200 (200 MHz for 1H and 50.3 MHz
for 13C) and Bruker AC 300 (300 MHz for 1H, 75.5 MHz for
13C and 96 MHz for 11B) spectrometers operating in the Fourier
transform mode. 13C NMR spectra were obtained with broad-
band proton decoupling. Chemical shifts are expressed as δ
values with tetramethylsilane or CCl3F (for 19F NMR) as
internal standard. For 11B NMR, the chemical schifts are given
in parts per million relative to BF3·OEt2. High-resolution mass
spectra were obtained with a Varian MAT 311 mass spectrom-
eter (Centre Re´gional de Mesures Physiques de l’Ouest,
Rennes, France). Microanalyses were performed at the Central
Laboratory for Analysis (CNRS, Lyon, France). Silica gel
60F254 was used for column chromatography.
1. Ch em ica l Syn th eses. Gen er a l P r oced u r e of Im in e
F or m a tion . To a stirred solution of amine (11 mmol) and 3 Å
molecular sieves (2 g) in anhydrous Et2O (50 mL) was added
a solution of aldehyde (10 mmol) in anhydrous Et2O (30 mL).
The mixture was stirred at room temperature for 16 h, until
the imine formation was complete (monitored by TLC). After
filtration, the solvent was evaporated under vacuum to give
the desired product, which was used without further purifica-
tion.
1
dioxaborane; colorless oil; 98%; H NMR (200 MHz, CDCl3) δ
8.21 (s, 1H), 7.78 (d, J ) 8.0 Hz, 2H), 7.60 (d, J ) 8.0 Hz, 2H),
4.65 (br, 1H), 3.69 (s, 4H), 3.60-3.45 (m, 2H), 3.28-2.92 (m,
6H), 1.95-1.76 (m, 2H), 1.52-1.25 (m, 22H), 0.94 (s, 6H); 13
C
NMR (50 MHz, CDCl3) δ 160.6, 155.0, 154.6, 136.9, 133.3,
126.1, 78.2, 71.2, 58.2, 45.7, 44.3, 39.2, 30.8, 27.4, 26.4, 20.9;
11B NMR (96 MHz, CDCl3) δ 27.4.
Gen er a l P r oced u r e of Red u ction . NaBH4 (20 mmol) was
added in small portions to a solution of imine (10 mmol) in
dry ethanol (60 mL) at 0 °C. After being stirred for 4 h, the
mixture was concentrated under vacuum. The residue was
taken up in water (60 mL) and extracted with CH2Cl2 (3 × 60
mL). The organic layers were combined, dried over MgSO4,
filtered, and concentrated under vacuum. The residue was
purified by flash chromatography on silica gel (210-400 mesh).
Da ta for ter t-Bu tyl N-{4-{[(4-F lu or op h en yl)m eth yl]-
a m in o}bu tyl}ca r ba m a te (3b): prepared from 2b; colorless
oil; 85% (Rf ) 0.4, ethyl acetate/methanol, 1:1); 1H NMR (200
MHz, CDCl3) δ 7.28-7.13 (m, 2H), 7.00-6.82 (m, 2H), 5.23
(br, 1H), 3.67 (s, 2H), 3.25-3.15 (m, 2H), 2.65-2.50 (m, 2H),
1.59-1.28 (m, 14H); 13C NMR (50 MHz, CDCl3) δ 148.5, 130.1,
129.9, 115.7, 115.3, 77.9, 53.7, 49.3, 41.0, 28.8, 28.3, 27.8; 19
F
NMR (282 MHz, CDCl3) δ -116.509.
Da ta for ter t-Bu tyl N-{4-{{[4-(5,5-Dim eth yl[1,3,2]d ioxa -
b or in a n -2-yl)p h e n yl]m e t h yl}a m in o}b u t yl}ca r b a m a t e
(3c): prepared from 2c; colorless solid; 65% (Rf ) 0.2,
1
methanol); mp ) 112-113 °C; H NMR (200 MHz, CDCl3) δ

N-Benzylpolyamines in Cancer Therapy and Imaging
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3661
7.78 (d, J ) 8.0 Hz, 2H), 7.32 (d, J ) 8.0 Hz, 2H), 4.88 (br,
1H), 3.82 (s, 2H), 3.80 (s, 4H), 3.22-3.10 (m, 2H), 2.75-2.60
(m, 2H), 1.62-1.40 (m, 14H), 1.05 (s, 6H); 13C NMR (50 MHz,
CDCl3) δ 155.0, 141.7, 133.0, 128.4, 126.3, 114.6, 77.9, 71.2,
52.9, 47.8, 39.4, 30.8, 27.4, 26.8, 26.3, 20.9; 11B NMR (96 MHz,
CDCl3) δ 26.7; HRMS (EI) calcd for C21H35BN2O4 (M+) 390.2690,
found 390.2691.
(m, 24H); 13C NMR (50 MHz, CDCl3) δ 156.0, 139.4, 128.9,
128.2, 127.0, 78.9, 78.7, 58.8, 53.3, 52.3, 40.4, 39.8, 28.5, 28.4,
27.8, 26.6, 24.2. Anal. (C24H41N3O4) C, H, N.
Gen er a l P r oced u r e of Am in o-Gr ou p Dep r otection . A
solution of N-tert-butoxycarbonyl derivative (3 mmol) in etha-
nol (26 mL) was treated with a 12 M aqueous solution of
hydrochloric acid (6.4 mL) at room temperature overnight.
After removal of the solvent, the mixture was diluted in water
(10 mL) and washed with ether. The aqueous solution was
concentrated and dried under high vacuum.
Da ta for N-(4-Am in obu tyl)(p h en ylm eth yl)a m in e Di-
h yd r och lor id e (bz-p u t, 4a ): prepared from 3a ; colorless
solid; 95%; mp 251-253 °C; 1H NMR (200 MHz, D2O) δ 7.28-
7.17 (m, 5H), 3.97 (s, 2H), 2.93-2.68 (m, 4H), 1.60-1.42 (m,
4H); 13C NMR (50 MHz, D2O) δ 131.0, 130.2, 130.0, 129.6, 51.4,
46.7, 39.2, 24.3, 23.1; HRMS (EI) calcd for C11H18N2 (M+)
178.1469, found 178.1471.
Da ta for N-(4-Am in obu tyl)[(4-flu or op h en yl)m eth yl]-
a m in e Dih yd r och lor id e (4-F bz-p u t, 4b): prepared from 3b;
colorless solid; 92%; mp 266-268 °C; 1H NMR (200 MHz, D2O)
δ 7.47-7.34 (m, 2H), 7.20-7.05 (m, 2H), 4.14 (s, 2H), 3.09 (t,
J ) 6.9 Hz, 2H), 3.00 (t, J ) 7.4 Hz, 2H), 1.75-1.59 (m, 4H);
13C NMR (50 MHz, D2O) δ 132.5, 132.3, 116.7, 116.2, 50.7, 46.6,
39.1, 24.3, 23.0; 19F NMR (282 MHz, D2O) δ - 111, 106; HRMS
(EI) calcd for C11H17N2F (M+) 196.1376, found 196.1372. Anal.
(C11H19N2Cl2F) C, H, N.
Data for N-(4-Am in obu tyl){{4-(5,5-dim eth yl[1,3,2]dioxa-
bor in an -2-yl)ph en yl}m eth yl}am in e Dih ydr och lor ide (4c):
prepared from 3c; colorless solid; 90%; mp > 300 °C; 1H NMR
(200 MHz, D2O) δ 7.95 (d, J ) 8.0 Hz, 2H), 7.60 (d, J ) 8.0
Hz, 2H), 4.38 (s, 2H), 3.48 (s, 4H), 3.30-3.08 (m, 4H), 1.95-
1.78 (m, 4H), 0.97 (s, 6H); 13C NMR (50 MHz, D2O) δ 134.8,
133.5, 129.6, 68.4, 51.3, 46.8, 39.2, 36.7, 24.3, 23.1, 20.8.
Da ta for N-{3-[(4-Am in obu tyl)a m in o]p r op yl}(p h en yl-
m eth yl)a m in e Tr ih yd r och lor id e (N1-bz-sp d , 10a ): pre-
pared from 9a ; colorless solid; 95%; mp > 300 °C; 1H NMR
(200 MHz, D2O) δ 7.48-7.26 (m, 5H), 4.13 (s, 2H), 3.15-2.80
(m, 8H), 2.10-1.88 (m, 2H), 1.76-1.55 (m, 4H); 13C NMR (50
MHz, D2O) δ 130.8, 130.2, 130.1, 129.7, 51.6, 47.4, 44.8, 44.2,
39.2, 24.3, 23.1, 23.0; HRMS (EI) calcd for C14H26N3 (M+ + H)
236.2127, found 236.2137. Anal. (C14H28N3Cl3) C, H.
Da ta for N-{3-[(4-Am in obu tyl)a m in o]p r op yl}[(4-flu o-
r op h en yl)m eth yl]a m in e Tr ih yd r och lor id e (N1-4-F bz-sp d ,
10b): prepared from 9b; colorless solid; 93%; mp > 300 °C;
1H NMR (300 MHz, D2O) δ 7.52-7.43 (m, 2H), 7.26-7.12 (m,
2H), 4.21 (s, 2H), 3.20-2.95 (m, 8H), 2.15-2.00 (m, 2H), 1.82-
1.65 (m, 4H); 13C NMR (50 MHz, D2O) δ 132.5, 132.4, 116.7,
116.2, 50.9, 47.4, 44.8, 44.2, 39.1, 24.2, 23.1, 23.0; 19F NMR
(282 MHz, D2O) δ -110.988; HRMS (EI) calcd for C14H25N3F
(M+ + H) 254.2033, found 254.2032. Anal. (C14H27N3FCl3) C,
H.
Da ta for ter t-Bu tyl N-{3-[(P h en ylm eth yl)a m in o]p r o-
p y l}-N -{4-{[(t er t -b u t y lo x y )c a r b o n y l]a m in o }b u t y l}-
ca r ba m a te (9a ): prepared from 8a ; colorless oil; 79% (Rf )
1
0.3, ethyl acetate/methanol, 1:1); H NMR (300 MHz, CDCl3)
δ 7.37-7.15 (m, 5H), 4.65 (br, 1H), 3.75 (s, 2H), 3.32-3.00 (m,
6H), 2.58 (t, J ) 6.9 Hz, 2H), 1.80-1.32 (m, 25H); 13C NMR
(50 MHz, CDCl3) δ 155.0, 154.5, 139.2, 127.4, 127.2, 125.9,
78.3, 53.0, 45.6, 39.2, 27.5, 27.4, 26.4.
Da ta for ter t-Bu tyl N-{3-{[(4-F lu or op h en yl)m eth yl]-
a m in o}p r op yl}-N-{4-{[(ter t-b u t yloxy)ca r b on yl]a m in o}-
bu tyl}ca r ba m a te (9b): prepared from 8b; colorless oil; 85%
(Rf ) 0.4, ethyl acetate/methanol, 1:1); 1H NMR (200 MHz,
CDCl3) δ 7.28-7.15 (m, 2H), 6.97-6.83 (m, 2H), 4.62 (br, 1H),
3.66 (s, 2H), 3.25-2.98 (m, 6H), 2.58-2.45 (m, 2H), 1.75-1.28
(m, 25H); 13C NMR (50 MHz, CDCl3) δ 155.0, 154.6, 135.0,
128.7, 128.6, 114.3, 113.9, 78.3, 52.3, 45.5, 39.2, 27.5, 27.4, 26.4;
19F NMR (282 MHz, CDCl3) δ -116.523.
Da ta for ter t-Bu tyl N-{3-{{[4-(5,5-Dim eth yl[1,3,2]d ioxa -
bor in a n -2-yl)p h en yl]m eth yl}a m in o}p r op yl}-N-{4-{[(ter t-
b u t yloxy)ca r b on yl]a m in o}b u t yl}ca r b a m a t e (9c): pre-
pared from 8c; colorless oil; 65% (Rf ) 0.2, methanol); 1H NMR
(200 MHz, CDCl3) δ 7.68 (d, J ) 8.0 Hz, 2H), 7.20 (d, J ) 8.0
Hz, 2H), 4.70 (br, 1H), 3.69 (s, 2H), 3.67 (s, 4H), 3.23-2.92
(m, 6H), 2.51 (t, J ) 6.7 Hz, 2H), 1.70-1.22 (m, 25 H), 0.94 (s,
6H); 13C NMR (50 MHz, CDCl3) δ 155.0, 154.6, 141.8, 133.0,
126.3, 78.2, 71.2, 53.1, 45.6, 39.2, 30.8, 27.5, 27.4, 26.4, 20.9;
11B NMR (96 MHz, CDCl3) δ 26.5. Anal. (C29H50BN3O6) C, H.
Gen er a l P r oced u r e of Alk yla tion . To a solution of 12
(43.4 mmol) in dry tetrahydrofuran/dimethyl sulfoxide (120
mL/30 mL) were added the bromide derivative (34.7 mmol),
NaHCO3 (86.8 mmol), and NaI (13 mmol). The mixture was
heated at reflux for 6 h. After removal of the tetrahydrofuran,
the mixture was diluted with ethyl acetate and washed with
H2O. MgSO4 was added, and filtration was followed by the
evaporation of the solvent. The residue was purified by flash
chromatography on silica gel (210-400 mesh).
Da ta for ter t-Bu tyl N-{4-{N-{3-[(ter t-Bu tyloxyca r bo-
n yl)a m in o]p r op yl}{[4-(5,5-d im eth yl[1,3,2]d ioxa bor in a n -
2-yl)p h en yl]m eth yl}a m in o}bu tyl}ca r ba m a te (14a ): pre-
pared from 12 and 2-[4-(bromomethyl)phenyl]-5,5-dimethyl-
1,3,2-dioxaborane (13a ); yellow oil; 51% (Rf ) 0.5 heptane/ethyl
acetate, 8:2); 1H NMR (200 MHz, CDCl3) δ 7.74 (d, J ) 7.9
Hz, 2H), 7.28 (d, J ) 7.9 Hz, 2H), 5.40 (br, 1H), 4.70 (br, 1H),
3.76 (s, 4H), 3.51 (s, 2H), 3.15-2.98 (m, 4H), 2.52-2.37 (m,
4H), 1.62-1.47 (m, 6H), 1.44 (s, 18H), 1.02 (s, 6H); 13C NMR
(50 MHz, CDCl3) δ 156.0, 141.9, 133.9, 128.2, 78.0, 77.9, 72.2,
58.9, 53.8, 52.7, 40.8, 40.1, 31.8, 28.5, 28.4, 27.8, 26.6, 24.2,
21.9; HRMS (EI) calcd for C29H50BN3O6 (M+) 547.3792, found
547.3794.
Da ta for N-{3-[(4-Am in obu tyl)a m in o]p r op yl}{{4-(5,5-
d im eth yl[1,3,2]d ioxa bor in a n -2 -yl)p h en yl}m eth yl}a m in e
Tr ih yd r och lor id e (10c): prepared from 9c; colorless solid;
90%; mp > 300 °C; 1H NMR (300 MHz, D2O) δ 7.30 (d, J ) 8.5
Hz, 2H), 6.88 (d, J ) 8.5 Hz, 2H), 4.13 (s, 2H), 3.32 (s, 4H),
3.15-2.91 (m, 8H), 2.16-2.00 (m, 2H), 1.80-1.65 (m, 4H), 0.82
(s, 6H); HRMS (EI) calcd for C19H34BN3O2 (M+) 347.2744, found
347.2748.
Da ta for ter t-Bu tyl N-{4-{N-{3-[(ter t-Bu tyloxyca r bo-
n yl)a m in o]p r op yl}{[3-(5,5-d im eth yl[1,3,2]d ioxa bor in a n -
2-yl)p h en yl]m eth yl}a m in o}bu tyl}ca r ba m a te (14b): pre-
pared from 12 and 2-[3-(bromomethyl)phenyl]-5,5-dimethyl-
1,3,2-dioxaborane (13b); yellow oil; 58% (Rf ) 0.5 heptane/ethyl
acetate, 8:2); 1H NMR (200 MHz, CDCl3) δ 7.70-7.20 (m, 4H),
5.33 (br, 1H), 4.75 (br, 1H), 3.76 (s, 4H), 3.52 (s, 2H), 3.18-
2.92 (m, 4H), 2.51-2.35 (m, 4H), 1.72-1.28 (m, 24H), 1.00 (s,
6H); 13C NMR (50 MHz, CDCl3) δ 156.1, 134.5, 132.8, 127.7,
78.0, 77.9, 72.3, 58.7, 53.2, 52.7, 40.8, 40.1, 31.9, 28.5, 28.4,
27.8, 26.6, 24.2, 21.9; HRMS (EI) calcd for C29H50BN3O6 (M+)
547.3792, found 547.3794.
Da ta for N-(3-Am in obu tyl)-N-(3-a m in op r op yl){{4-(5,5-
d im e t h y l[1,3,2]d io x a b o r in a n -2-y l)p h e n y l}m e t h y l}-
a m in e Tr ih yd r och lor id e (N4-4-Bbz-sp d , 15a ): prepared
1
from 14a ; amorphous solid; 92%; H NMR (300 MHz, D2O) δ
7.83 (d, J ) 8.1 Hz, 2H), 7.52 (d, J ) 8.1 Hz, 2H), 4.42 (s, 2H),
3.34 (s, 4H), 3.28-3.18 (m, 4H), 3.10-2.95 (m, 4H), 2.26-2.05
(m, 2H), 1.81-1.64 (m, 4H), 0.82 (s, 6H); 13C NMR (50 MHz,
D2O) δ 134.9, 131.4, 130.7, 68.7, 57.5, 52.3, 49.9, 39.1, 36.9,
36.3, 24.2, 22.0, 20.8, 20.6; 11B NMR (96 MHz, D2O) δ 29.5;
HRMS (EI) calcd for
347.2748.
C
19H34BN3O2 (M+) 347.2744, found
Data for ter t-Bu tyl N-{4-{N-{3-[(ter t-Bu tyloxycar bon yl)-
a m i n o ]p r o p y l}(p h e n y lm e t h y l)a m i n o }b u t y l}c a r b a -
m a te56 (14c): prepared from 12 and benzyl bromide; yellow
Da ta for N-(3-Am in obu tyl)-N-(3-a m in op r op yl){{3-(5,5-
d im e t h y l[1,3,2]d io x a b o r in a n -2-y l)p h e n y l}m e t h y l}-
a m in e Tr ih yd r och lor id e (N4-3-Bbz-sp d , 15b): prepared
1
oil; 70% (Rf ) 0.5, heptane:ethyl acetate, 9:1); H NMR (200
MHz, CDCl3) δ 7.35-7.20 (m, 5H), 5.48 (br, 1H), 4.80 (br, 1H),
3.50 (s, 2H), 3.25-3.00 (m, 4H), 2.52-2.33 (m, 4H), 1.70-1.41
1
from 14b; amorphous solid; 90%; H NMR (300 MHz, D2O) δ

3662 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Martin et al.
7.92-7.50 (m, 4H), 4.44 (s, 2H), 3.35 (s, 4H), 3.36-3.20 (m,
4H), 3.10-2.95 (m, 4H), 2.30-2.15 (m, 2H), 1.95-1.60 (m, 4H),
0.83 (s, 6H); HRMS (EI) calcd for C19H34BN3O2 (M+) 347.2744,
found 347.2748.
not shown), their culture in the presence of calf serum did not
require the addition of aminoguanidine, an inhibitor of this
enzyme.
c. In Vitr o Eva lu a tion of Dr u g Cytotoxicity/Cytosta sy.
The effect of the polyamine derivatives on cell growth was
assayed in 96-well plates (Becton Dickinson, Oxnard, CA).
Twenty-four hours after seeding (initial density 1000 3LL cells/
well), drugs were added in culture medium. Cell growth rates
were determined by measuring formazan formation from
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) using a Titertek Multiskan MCC/340 microreader
(Labsystems, Life Sciences Interactions, Cergy-Pontoise, France)
for absorbance measurements.58
d . Cellu la r Up ta k e. Cells were cultured in tissue culture
flasks with initial densities of 5 × 104 cells/mL. Unless stated
otherwise, drugs were added 24 h after seeding. Cells were
harvested using trypsine/EDTA. All cells were washed three
times with phosphate-buffered saline before processing.
e. Det er m in a t ion of P olya m in es a n d of P olya m in e
Der iva tives. Cells were disrupted by sonication in 0.2 N
perchloric acid and proteins precipitated by centrifugation at
1500g. Each supernatant was frozen at -20 °C until analysis
by HPLC. The protein content was determined using the Folin
phenol reagent.59 Polyamines and their derivatives were
determined by separation of the ion pairs formed with n-
octanesulfonic acid (for polyamines and all derivatives except
for 4-F bz-p u t) or n-hexanesulfonic acid (for 4-F bz-p u t) on a
reversed-phase column, reaction of the column effluent with
o-phthalaldehyde and N-acetylcysteine, and monitoring of
fluorescence intensity as previously described.60
f. Bor on Mea su r em en ts. Capped Teflon digestion bombs
containing the cells in 2 mL of 60% nitric acid were heated
for a 90 min cycle using a microwave oven (MDS-8ID, CEM,
Mattews; maximal power 630 ( 70 W). After cooling, 2 mL of
30% hydrogen peroxide was added. After another heating cycle,
the samples were transferred to polypropylene tubes and
completed to 5 mL with Millipore water. Boron standards were
prepared from a boron atomic absorption standard solution
(Sigma) and diluted in a mixture of nitric acid, hydrogen
peroxide, and water (2:2:1). ICP-AES determinations of boron
were done using the 249.773 nm line on an ICP atomic
emission spectrometer (J Y 38 S, J obin-Yvon ISA, Longjumeau,
France).
Da ta for N-(3-Am in obu tyl)-N-(3-a m in op r op yl)(p h en yl-
m eth yl)a m in e Tr ih yd r och lor id e (N4-4-bz-sp d , 15c): pre-
1
pared from 14c; amorphous solid; 95%; H NMR (200 MHz,
D2O) δ 7.42-7.28 (m, 5H), 4.26 (s, 2H), 3.20-3.00 (m, 4H),
2.96-2.80 (m, 4H), 2.15-1.42 (m, 6H); 13C NMR (50 MHz, D2O)
δ 131.4, 130.8, 129.3, 129.1, 57.8, 52.4, 49.9, 39.2, 37.0, 24.3,
22.1, 21.0; HRMS (EI) calcd for C14H25N3 (M+) 235.2048, found
235.2050.
Gen er a l P r oced u r e for th e Clea va ge of Bor on ic Ester .
To a stirred solution of the boronic ester (1 mmol) in water
(15 mL) was added phenylboric acid (1 mmol) and ether (15
mL). After 3 h at room temperature the ether layer was
removed and replaced by fresh ether (15 mL). After a further
3 h, the ether layer was again removed and replaced. The
mixture was then stirred for an additional 3 h. The ether layer
was removed, and the aqueous phase was washed with ether.
The aqueous solution was concentrated and dried under high
vacuum.
Data for N-(4-Am in obu tyl){[4-(dih ydr oxybor yl)ph en yl]-
m eth yl}a m in e Dih yd r och lor id e (4-Bbz-p u t, 5): prepared
1
from 4c; colorless solid; 95%; mp 262-264 °C; H NMR (200
MHz, D2O) δ 7.88 (d, J ) 8.0 Hz, 2H), 7.55 (d, J ) 8.0 Hz,
2H), 4.30 (s, 2H), 3.12 (t, J ) 6.8 Hz, 2H), 3.05 (t, J ) 6.7 Hz,
2H), 1.90-1.70 (m, 4H); 13C NMR (50 MHz, D2O) δ 134.7,
133.4, 129.6, 51.3, 47.4, 39.2, 24.3, 23.1; 11B NMR (96 MHz,
CDCl3) δ 29.3; HRMS (EI) calcd for C25H30BN4O6 with matrix
3-nitrobenzyl alcohol (M+ + H) 493.2258, found 493.2262. Anal.
(C11H21BN2O2Cl2) C, H, N.
Da ta for N-{3-[(4-Am in obu tyl)a m in o]p r op yl}{[4-(d ih y-
d r oxybor yl)p h en yl]m eth yl}a m in e Tr ih yd r och lor id e (N1-
4-Bbz-sp d , 11): prepared from 10c; colorless solid; 93%; mp
> 300 °C; 1H NMR (300 MHz, D2O) δ 7.80 (d, J ) 7.9 Hz, 2H),
7.45 (d, J ) 7.9 Hz, 2H), 4.25 (s, 2H), 3.22-2.95 (m, 8H), 2.18-
2.03 (m, 2H), 1.80-1.66 (m, 4H); 13C NMR (50 MHz, D2O) δ
134.8, 133.2, 129.6, 51.5, 47.4, 44.8, 44.4, 39.2, 24.3, 23.1, 23.0;
11B NMR (96 MHz, D2O) δ 28.5; HRMS (EI) calcd for C28H37
-
BN5O6 with matrix 3-nitrobenzyl alcohol (M+ + H) 550.2837,
found 550.2840.
2. Biologica l Stu d ies. Unless otherwise stated, the usual
laboratory chemicals were purchased from Merck (Darmstadt,
Germany) or Sigma Chemical Co. (St. Louis, MO). DFMO was
obtained from Ilex Oncology (San Antonio, TX). BPA and BSH
were purchased from Boron Biologicals Inc. (Raleigh, NC).
Data are given as mean values and SD (or SEM) of three or
more experiments. Comparisons between means were made
using the Student’s t-test assuming significance at p < 0.01.
a . Eth id iu m Br om id e Disp la cem en t Assa y. The release
of ethidium bromide from DNA was monitored fluorimetrically
as previously described57 using a Fluorolog 2 spectrofluorim-
eter (J obin-Yvon ISA, Longjumeau, France) at excitation and
emission wavelengths of 546 and 595 nm, respectively. Calf
thymus DNA (0.2 A260 unit) was added to 3 mL of buffer (5
mM NaCl/1 mM sodium cacodylate, pH 7) containing 2.0 µM
ethidium bromide. The complex was titrated by adding µL
volumes of concentrated solutions of polyamines or polyamine
derivatives at room temperature. The fluorescence intensity
was corrected for the dilution.
b. Cell Cu ltu r e. Murine 3LL Lewis lung carcinoma, murine
B16 melanoma, rat C6 glioblastoma, human U251 glioblas-
toma (obtained from the brain tumor tissue bank at the
Department of Neurosurgery, University of California, San
Francisco), CHO, and CHO-MGR (provided by Dr. W. Flintoff,
University of Western Ontario) cells were grown in RPMI 1640
medium (Eurobio, Les Ulis, France) supplemented with 10%
fetal calf serum, 2 mM glutamine, streptomycin (50 µg/mL),
and penicillin (100 U/mL) (Biomerieux, Marcy l’Etoile, France).
A 2 µg/mL sample of L-proline was added to the culture
medium for CHO-MGR cells. Cells were grown at 37 °C under
a humidified 5% CO2 atmosphere. Because none of the drugs
tested were substrates of the bovine serum amine oxidase (data
Ack n ow led gm en t. We are indebted to Pr. W. Flint-
off (University of Western Ontario, London, Canada) for
providing the CHO-MG cells. We acknowledge the
Ministe`re de la Recherche et de la Technologie (MRT),
the Centre National de la Recherche Scientifique (CNRS),
the Association pour la Recherche sur le Cancer (ARC),
the Ligue Nationale Contre le Cancer (LNCCsComite´s
d’Ille-et-Vilaine, du Morbihan et des Coˆtes d’Armor), and
the Conseil Re´gional de Bretagne (CRB) for financial
support and for fellowships to F.P. (CRB), B.M. (LNCC
and ARC), and C.L. (MRT). Our sincere thanks are also
due to D. Desplanques and A. Papillon (Laboratoire
Interre´gional de la DGCCRF, Rennes, France) for giving
us access to the ICP-atomic emission spectrometer as
well as for their appreciable advice and technical
assistance. We also gratefully acknowledge R. Havouis
for the HPLC determinations.
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N-Benzylpolyamines in Cancer Therapy and Imaging
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