Effect of spermine conjugation on the cytotoxicity and cellular transport of acridine

Article abstract of DOI:10.1021/jm020843w

Polyamines are believed to be potent vectors for the selective delivery of chemotherapeutic agents into cancer cells. In this paper, we report the effect of spermine conjugation on the cytotoxic and transport properties of acridine. Six derivatives, composed of a spermine chain attached at its N¹ position to an acridine via an aliphatic chain, were synthesized. The aliphatic linker, comprised of 3-5 methylene units, was connected to the position-9 of the heterocycle through either an amide (amidoacridines 8-10) or an amine (aminoacridines 11-13) linkage. Independently of their architecture, all ligands showed a high affinity for DNA binding but a limited DNA sequence selectivity. In a whole cell assay with L1210 and Chinese hamster ovary (CHO) cells, the aminoacridines (IC₅₀ values around 2 μM) were more potent than the amidoacridines (IC₅₀ values between 20 and 40 μM). This was related to a less efficient transport for the latter. As determined from competitive uptake studies with [¹⁴C] spermidine, all conjugates had a high affinity for the polyamine transport system (PTS). However, on the basis of competitive studies with an excess of spermidine and on the differential effect on cell growth and accumulation in CHO and in the mutant PTS deficient CHO-MG cells, the accumulation of the conjugates through the PTS was found to be poor but still more efficient for the aminoacridines. α-Difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, which induces an up-regulation of the activity of the PTS, enhanced accumulation of all acridine conjugates through the PTS and had a synergistic effect on the potency of the acridine conjugates to inhibit cell growth. Despite their high affinity for the PTS, the low amount of derivatives transiting through the PTS is likely to be related to their ability to repress rapidly and efficiently the activity of the PTS and, consequently, to inhibit their own uptake via this system.

Full text of DOI:10.1021/jm020843w

5098
J . Med. Chem. 2002, 45, 5098-5111
Effect of Sp er m in e Con ju ga tion on th e Cytotoxicity a n d Cellu la r Tr a n sp or t of
Acr id in e
J ean-Guy Delcros,*,† Sophie Tomasi,‡ Simon Carrington,§ Be´ne´dicte Martin,† J acques Renault,‡
Ian S. Blagbrough,§ and Philippe Uriac‡
Groupe de Recherche en The´rapeutiques Anticance´reuses, UPR ESA CNRS 6027, Faculte´ de Me´decine, UPRES EA 2234,
Faculte´ de Pharmacie, Universite´ Rennes 1, 2 Avenue du Professeur Le´on Bernard, 35043 Rennes Ce´dex, France, and
Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, United Kingdom
Received February 4, 2002
Polyamines are believed to be potent vectors for the selective delivery of chemotherapeutic
agents into cancer cells. In this paper, we report the effect of spermine conjugation on the
cytotoxic and transport properties of acridine. Six derivatives, composed of a spermine chain
attached at its N1 position to an acridine via an aliphatic chain, were synthesized. The aliphatic
linker, comprised of 3-5 methylene units, was connected to the position-9 of the heterocycle
through either an amide (amidoacridines 8-10) or an amine (aminoacridines 11-13) linkage.
Independently of their architecture, all ligands showed a high affinity for DNA binding but a
limited DNA sequence selectivity. In a whole cell assay with L1210 and Chinese hamster ovary
(CHO) cells, the aminoacridines (IC50 values around 2 µM) were more potent than the
amidoacridines (IC50 values between 20 and 40 µM). This was related to a less efficient transport
for the latter. As determined from competitive uptake studies with [14C]spermidine, all
conjugates had a high affinity for the polyamine transport system (PTS). However, on the basis
of competitive studies with an excess of spermidine and on the differential effect on cell growth
and accumulation in CHO and in the mutant PTS deficient CHO-MG cells, the accumulation
of the conjugates through the PTS was found to be poor but still more efficient for the
aminoacridines. R-Difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase,
which induces an up-regulation of the activity of the PTS, enhanced accumulation of all acridine
conjugates through the PTS and had a synergistic effect on the potency of the acridine conjugates
to inhibit cell growth. Despite their high affinity for the PTS, the low amount of derivatives
transiting through the PTS is likely to be related to their ability to repress rapidly and efficiently
the activity of the PTS and, consequently, to inhibit their own uptake via this system.
In tr od u ction
high demand for polyamines such as tumors take up
radiolabeled polyamines in greater amounts than other
tissues.10,11 As the polyamine content of tumor cells is
more efficiently depleted by DFMO than that of non-
or slow-growing cells, DFMO increases the uptake rate
of tumor cells that readily accumulate high amounts of
polyamines.10-13
Natural polyamines, such as spermine (1) and sper-
midine (2) are ubiquitous polycationic molecules, which
play crucial roles in a number of cell processes including
cell proliferation and differentiation.1 In eukaryotic cells,
intracellular polyamine pool size is determined by
highly regulated complex biosynthetic and catabolic
pathways as well as by specific active transport systems
allowing polyamine exchange (uptake and release) with
the environment. Intracellular levels of free polyamines
control the activity of the uptake system as well as the
anabolic pathways.2-4 Structural features of polyamines,
the high need of tumor cells for these polycations, along
with the characteristics of their cell transport systems
make polyamines attractive vectors for tumor target-
ing.4 The activity of the polyamine transport system
(PTS) is higher in highly proliferating cells such as
tumor cells than in resting cells.5,6 Depletion of intra-
cellular polyamine pools, e.g., by R-difluoromethyl-
ornithine (DFMO), a suicide substrate inhibitor of
ornithine decarboxylase, greatly enhances polyamine
uptake by various cell lines.7-9 In vivo, tissues with a
The selectivity of the PTS is not restricted to natural
polyamines. Indeed, polyamine uptake systems have a
low stringency for structural features thereby allowing
a wide variety of synthetic polyamine analogues to be
taken up by these systems.4 The PTS may therefore
afford an alternative mode of entry into cells for more
selective chemotherapeutics. The vectorization of che-
motherapeutic drugs by polyamines is expected to
enhance cytotoxicity to tumor cells and diminish sec-
ondary effects on normal cells. These characteristics
have led to the design of polyamine conjugated to known
chemotherapeutic drugs. For example, chlorambucil,14-16
nitroimidazole,17 and aziridine18-20 polyamine conju-
gates have been synthesized with improved therapeutic
indexes. However, evidence concerning the ability of
these conjugates to use the PTS is mostly based on
indirect arguments: (i) their ability to inhibit the
transport of radiolabeled polyamines, (ii) the synergistic
effect of DFMO, or (iii) reduced cytotoxicity in the pres-
ence of exogenous polyamines. However, impermeant
* To whom correspondence should be addressed. Tel: 33(0)2.23.
23.45.03. Fax: 33(0)2.23.23.46.08. E-mail: delcros@univ-rennes1.fr.
† Faculte´ de Me´decine, Universite´ Rennes 1.
‡ Faculte´ de Pharmacie, Universite´ Rennes 1.
§ University of Bath.
10.1021/jm020843w CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/02/2002

Effect of Spermine Conjugation on Acridine
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5099
polyamine analogues with high affinity for the polyamine
transport have been reported.21 On the other hand,
bis(benzyl)polyamine analogues inhibit polyamine up-
take but are substrates for a cell transport system
distinct from the PTS.22,23 In addition, the cytotoxicity
of therapeutic drugs unrelated to polyamines, such as
BCNU, is enhanced by DFMO.24 Recent studies with
fluorescein-polyamine conjugates have however sug-
gested a broad structural tolerance, the initial velocity
of transport of these conjugates being similar to that of
the free polyamine homologues.25
Among many targets, DNA has been considered as a
choice target for anticancer strategy. Disrupting DNA
functions or structure will block cell proliferation or kill
cells. Numerous DNA intercalating or groove binding
agents are potent cytotoxic drugs.26 Because of the lack
of specificity of the target, the activity of DNA targeting
drugs may be improved by increasing their selectivity
for tumor cells as well as their affinity for DNA.
Tethering DNA targeting drugs to polyamines may
respond to both imperatives. SPM and SPD have a high
affinity for DNA, and despite a limited DNA sequence
selectivity,27 they may be involved in vivo in DNA
conformational changes and chromatin condensation.28
Coupling a polyamine to a DNA targeting agent is
believed to increase its DNA binding affinity through
electrostatic interactions. The conjugation of the aro-
matic nitrogen mustard chlorambucil, a clinically im-
portant chemotherapeutic agent, to SPD enhances by a
factor close to 104 its efficiency of cross-linking without
altering its preferred cross-linking site.14,29 The presence
of a polyamine side chain on intercalating agents such
as anthracene or acridine (3 and 4) does not prevent
the intercalating capability of the drug although the
geometry of the complex is slightly altered, and also, it
does allow the conjugate to have higher affinity by using
additional nonintercalative binding modes.30-32 It has
also been shown that polyamine conjugation does not
disrupt the topoisomerase II inhibitory activity of acri-
dine.33,34 Taken together, these data suggest that teth-
ering a polyamine chain to a DNA binding agent will
not alter and possibly may improve its activity.
Recently, Phanstiel and collaborators33,34 raised the
question as to whether polyamine conjugation to bulky
DNA binding agents such as linear tricyclic systems (5
and 6) may improve their selectivity by targeting the
PTS. On the basis of a SPD protection assay, it was
concluded that the monoalkylated SPM motif present
in 6 was a better vector than its SPD homologue 5. This
same issue is further addressed in the present work.
Our novel analogues are composed of a SPM covalently
bound to an acridine via an aminoaliphatic linker. The
linker was attached to the 9-position of acridine, through
either an amide bond (amidoacridines 8-10) or directly
as the aniline (aminoacridines 11-13). The architecture
of the conjugates was based on the following observa-
tions: (i) Because N1 derivatives of polyamines show a
higher affinity for the polyamine transporter than their
N4 homologues,35-38 the SPM moiety was linked through
one of its terminal amino groups. (ii) Earlier studies
demonstrated a relationship between the number of
protonated nitrogens along polyamines and their de-
rivatives, and their affinity for the PTS35,39,40 as well
as for DNA.27 To preserve the full cationic charge of
F igu r e 1. Structures of spermine (1), spermidine (2), Blag-
brough’s conjugates (3 and 4), Phanstiel’s conjugates (5-7),
and our acridine-spermine conjugates (8-13).
SPM, the linker was bound to SPM via an amine bond.
In the present work, conjugates 8-13 were studied for
their DNA binding properties, cell growth inhibitory
effects, and their interactions with the PTS.
Ch em ica l Syn th eses
The SPM-acridine conjugates 8-13 (Figure 1) differ
in two main characteristics: the bond (amide or amine)
and the length of the spacer (3-5 methylenes) between
the acridine and the SPM moieties. They were obtained
by conjugation of three unsymmetrically protected SPM
derivatives to acridine-9-carboxylic acid or 9-phenoxy-
acridine.
As shown in Scheme 1, the synthesis began by the
Swern oxidation41-43 of the N-CBZ-3-aminopropan-1-ol
14 affording the desired aldehyde 17 in 50% yield. The
oxidation of N-CBZ-4-aminobutan-1-ol 15 and of N-CBZ-
5-aminopentan-1-ol 16 led to the unexpected N-CBZ-
cyclic aminals 18 and 19 in 87 and 73%, respectively.
The cyclic structures were assessed by 13C NMR spec-
troscopic data: we observed two signals at 81.2 and 82.0
ppm (mixture of conformational isomers) for compound
18. These chemical shifts could not be assigned to an
aldehydic carbon (201.22 ppm for 17). They are de-
scribed as equivalent to the corresponding aminoalde-
hyde because of their existence in equilibrium with the
required aldehyde.44-48 To our knowledge, this is the
first report of the generation of these aminals by
oxidation of the corresponding amino alcohols. The
existence of this equilibrium was proved by the suc-
cessful reductive alkylation of 18 and 19 with the
N1,N,4N9-tri-tert-butoxycarbonylspermine (H2NA ) 20).49
They reacted in MeOH in the presence of NaBH3CN.
The pH of the reaction was maintained between 6.0 and
7.5 by the dropwise addition of glacial AcOH. The
unsymmetrical polyamine moieties 22 and 23 were
obtained after 48 or 72 h in 40 and 29%, respectively.
The reductive amination of 20 with the aminoaldehyde

5100 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Delcros et al.
Sch em e 1a
a
Reagents: (a) DMSO, (COCl)2, Et3N, anhydrous CH2Cl2, -78 °C. (b) NaBH3CN, glacial acetic acid, 4 Å molecular sieves, anhydrous
MeOH. (c) (Boc)2O, DMF and then H2, 1 atm, Pearlman’s catalyst, MeOH.
Sch em e 2a
respectively, by alkylation of the polyamines 24-26
with the previously prepared 9-phenoxyacridine.50-52
The target hydrochlorides 8-13 were then generated
in good yields using a 0.9 M solution of HCl gas in ethyl
acetate.
Biologica l Stu d ies
1. Op tica l P r op er ties. All six acridine conjugates
absorbed light between 350 and 450 nm and emitted
fluorescence. The characteristics of their absorption and
fluorescence spectra were (i) dependent on the nature
of the bond (amine or amide) between the linker and
the acridine heterocycle, (ii) insensitive to the length of
the tether, and (iii) modified upon binding to DNA. As
an illustration, absorption and fluorescence excitation
and emission spectra of conjugates 10 and 11 in the
absence and presence of calf thymus DNA (CT-DNA)
are shown in Figure 2. Free amidoacridines showed a
maximal absorption at 359 nm while two absorption
bands (λmax ) 410 and 430 nm) were observed for the
aminoacridines. Fluorescence spectra of the amido-
acridines at pH 7 showed an excitation spectrum
centered at 359 nm and a maximum emission at 435
nm. In acidic medium (0.2 N HClO4), the excitation
spectra showed two bands (λmax ) 360 and 410 nm) and
the emission maximum was shifted to 492 nm. For the
aminoacridines, emission maxima at 449 and 466 nm
were seen at pH 7 as well as in the acidic medium (data
not shown). In the presence of CT-DNA, spectral changes
were observed as reported for other acridine de-
rivatives.53,54 This involved a hypochromic effect and
bathochromic shifts (λmax ) 362 nm for the amido-
acridines; λmax ) 415 and 440 nm for the aminoacridines)
with clear isosbestic points observed at 400 and 445 nm
for the aminoacridines and amidoacridines, respectively.
Bathochromic shifts clearly sign the binding of indi-
vidual ligands to the DNA substrate, and isosbestic
points are indicative of equilibrium between bound and
free molecules. Hypochromism and decrease in the
fluorescence emission intensity in the presence of CT-
DNA indicate that the DNA-bound acridine derivatives
are in a less polar environment.30,31,55,56
a
Reagents: (a)
DCC, HOBt, DMF. (b) 0.9 M HCl/EtOAc. (c)
PhOH, ∆.
17 was faster (24 h) and gave the required polyamine
21 in a higher yield (54%).
The last step before the condensation with acridine
derivatives was the BOC protection of the free amino
group followed by the removal of the CBZ group by
Pearlman’s catalyst under H2. We obtained the desired
BOC-protected polyamines 24-26 in 63, 32, and 34%,
respectively.
As shown in Scheme 2, the coupling between pro-
tected polyamine and acridine moieties involved two
different routes. The acylation of 24-26 with acridine-
9-carboxylic acid was mediated by DCC and a catalytic
amount of N1-hydroxybenzotriazole affording the com-
pounds 27-29 in 64, 54, and 67%, respectively. The
compounds 30-32 were obtained in 65, 54, and 59%,

Effect of Spermine Conjugation on Acridine
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5101
F igu r e 2. Absorption (upper panels) and fluorescence excitation and emission spectra (lower panels) of the conjugates 10 (left
panels) and 11 (right panels). Upper panels: Visible absorption spectra of 10 µM solution of the conjugates in 10 mM Tris-HCl
(pH 7.4), 50 mM NaCl in the absence (a) or in the presence of 100 µM CT-DNA (b). Lower panels: Fluorescence excitation (c and
f) and emission (d, e, and g) spectra of 0.25 µM solutions of the conjugates either in 10 mM Tris-HCl (pH 7.4), 50 mM NaCl in the
absence (c and d) or in the presence of 30 µM CT-DNA (e) or in 0.2 N HClO4 (f and g).
Ta ble 1. DNA Binding Dataa
be 107 M-1 57
,
a Kapp of approximately 1.1 × 108 M-1 can
Q (µM)b
be calculated for the derivatives. This compares with
1.9 × 106 M-1 for SPM and 8.9 × 104 M-1 for acridine.58
Thus, tethering SPM to acridine increased considerably
the overall DNA binding affinity as expected from the
synergistic activity of two ligands with DNA affinity.30
The length of the tether as well as the nature of the
bond connecting the tether to the acridine moiety had
compd
AT
CT-DNA
GC
CC50 (µM)c
8
9
10
11
12
1.7
1.8
1.9
1.8
1.9
1.7
nde
26
1.8
1.9
2.0
2.0
2.0
1.8
nd
2.0
2.1
2.1
2.2
2.4
2.3
nd
0.12
0.12
0.12
0.11
0.11
0.11
6.8
13
no influence on the Kapp
.
SPM 1
acridined
24
22
142
Quenching fluorimetric assays with DNA-bound ethid-
ium (at a low [ethidium]:[DNA] molar ratio, e.g., 0.1:1)
can be used to distinguish intercalating agents from
nonintercalative ligands59 and also to determine possible
base or sequence preferential binding.60 The fluores-
cence quenching (Q) values with [poly(dA-dT)]2 are
much smaller than expected for the untethered DNA
intercalating acridine.58 The Q values are closer to
values reported for nonintercalative minor groove bind-
ing ligands such as distamycin A,60 which are more
efficient quenchers of the bound ethidium fluorophore
than planar intercalators because of their larger DNA
binding site sizes. In addition, the close Q values deter-
mined using CT-DNA, [poly(dA-dT)]2, and [poly(dG-
dC)]2 show that the acridine conjugates do not exhibit
any significant base selectivity, in contrast to the
preference for GC- and AT-rich sequences of acridine58
and spermine,27 respectively. Altogether, these results
indicate that groove binding modes are involved for
DNA binding of the SPM-acridine conjugates and that
the binding behavior is more directed by the polycationic
SPM side chain than by the intercalating chromophore,
as already reported for other acridine conjugates with
a
AT, CT-DNA, and GC refer to [poly(dA-dT)]2, calf thymus
b
DNA, and [poly(dG-dC)]2, respectively. Conjugate concentration
to give 50% fluorescence quenching of bound ethidium at pH 5 for
an [ethidium]:[DNA] ratio of 0.1:1; mean value ((10%) from three
determinations. c Drug concentration to give 50% drop in fluores-
cence of bound ethidium for an [ethidium]:[CT-DNA] ratio of 1.26:
d
1; mean value ((5%) from three determinations. Data taken from
ref 58. e nd, not determined.
2. DNA Bin d in g P r op er ties. “Apparent” equilibri-
um constants (Kapp) of drug binding to DNA can be
estimated by measuring the loss of ethidium fluores-
cence consecutive to the displacement of the DNA-bound
fluorochrome (at a high [ethidium]:[DNA] molar ratio,
e.g., 1.26:1) as a function of added drug. The drug
concentration producing 50% inhibition of fluorescence
(CC50) is approximately inversely proportional to the
binding constant.57 From Table 1, the CC50 values
determined for ethidium displacement from CT-DNA
were found to be around 0.11 µM for all six acridine
conjugates and 6.8 µM for free spermine. For untethered
acridine, a CC50 value of 142 µM has been previously
reported.58 Taking the binding constant of ethidium to

5102 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Delcros et al.
F igu r e 3. Effect of the conjugates 10 (left panels) and 11 (right panels) on the growth of L1210 (upper panels), CHO, and CHO-
MG (lower panels) cells in the presence or absence of DFMO. Cells were treated for 48 h with the conjugates in the presence or
absence of 5 mM DFMO. Cell growth rates were determined using an MTT assay. The relative cell growth rates were calculated
from the value of cell growth of the corresponding control cells cultured in the absence (OD540 nm ) 0.87, 0.79, and 0.94 for CHO,
CHO-MG, and L1210 cells, respectively) or in the presence of DFMO (OD540 nm ) 0.50, 0.45, and 0.55 for CHO, CHO-MG, and
L1210 cells, respectively).
functionalized side chains.58,60,61 This is also consistent
with the multiple binding modes observed with our
anthracene-polyamine conjugates.30-32
Ta ble 2. In Vitro Cell Growth Inhibitiona
inhibitory IC50/IC25 concn (µM)c
CHO
jugate control + DFMOb control + DFMOb control + DFMOb
L1210
CHO-MG
con-
3. Gr ow th In h ibitor y Effects a n d Up ta k e. As with
free SPM and other SPM derivatives containing a free
terminal primary amine,62,63 all six acridine conjugates
were substrates for bovine serum amine oxidase (data
not shown). To prevent cytotoxicity induced by the
oxidation products, all experiments were performed in
the presence of aminoguanidine (2 mM), an inhibitor of
the oxidase. In the presence of micromolar concentra-
tions of the conjugates, the growth rate of L1210 and
Chinese hamster ovary (CHO) cells decreased in a
concentration-dependent manner. Typical results are
shown in Figure 3, and IC50 values are listed in Table
2. The architecture of the conjugates clearly modulates
their bioactivity, and the nature of the chemical bond
linking the amino-aliphatic chain to the acridine moiety
has a profound impact on the toxicity of the derivatives.
The aminoacridines 11-13 (IC50 values around 2 µM)
were 10-20 times more potent than the amidoacridines
8-10 (IC50 values between 20 and 40 µM). The length
of the spacer showed little influence on the toxicity of
the drugs. Qarawi et al.52 have reported the cytotoxicity
on B16 melanoma cells of conjugates composed of a
spermine chain conjugated directly to the 9-position of
acridine through either an amide (3) or an amine (4)
8
9
43/22
18/10
2.2/0.09 30
2.2/0.16 17
ndd
nd
35
21
nd
nd
10
11
12
13
41/22
6.6/0.10 25/10.8 2.0/0.025 31/10.5
23/6.0
7.3/2.5
nd
2.8/1.4
2.3/1.1
1.4/0.4
0.9/0.14 2.3/0.9 1.3/0.32
6.1/1.8
7.2
0.6/0.16 2.3
0.7/0.20 1.0
nd
nd
4.0
nd
a
All cells were challenged with the conjugates at the time of
seeding for L1210 cells and 24 h later for CHO and CHO-MG cells.
Cell growth was determined using an MTT assay after 48 h
b
treatment. DFMO (5 mM) was added at the time of the addition
of the conjugates. c Conjugate concentration required to inhibit cell
growth by 50 or 25% after 48 h; mean value ((10%) from three
d
determinations. nd, not determined.
bond. The aminoacridine 4 was more toxic than the
amidoacridine 3. All of these data demonstrate that in
terms of cell growth effects, aminoacridines are more
favorable than amidoacridines. The positively charged
amino group on the 9-position of the aminoacridines is
likely to be a critical feature. The site of conjugation on
the spermine chain has also a profound impact on the
activity of the conjugates in terms of toxicity. Conjuga-
tion on the terminal amino group is more beneficial than

Effect of Spermine Conjugation on Acridine
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5103
Ta ble 3. Inhibition of [14C]SPD Uptake and Cellular Uptake of the Acridine Conjugates in L1210 Cells
cellular uptake in L1210 (nmol/mg protein)b
inhibition of [14C]SPD
concn
(µM)
500 µM
pretreated with
5 mM DFMOe
ratio (
DFMO
d
compd
uptake, Ki (µM)a
control
SPDc
∆
SPM 1
1.34
8
0.097
10
5
10
10
2
0.5
2
1
0.5
2
1
0.5
2
0.239
0.086
0.561
0.279
0.064
0.033
0.717
0.307
0.186
0.491
0.362
0.193
0.510
0.221
0.073
nd
0.262
0.049
0.016
0.553
0.188
0.053
nd
0.018
0.013
0.587
nd
2.4
9
10
0.083
0.097
0.850
0.631
0.217
0.113
1.583
0.728
0.496
1.528
nd
1.5
2.3
3.4
3.4
2.2
2.4
2.7
3.1
0.017
0.015
0.017
0.136
0.119
0.133
11
12
13
0.129
0.083
0.135
0.280
0.094
nd
0.082
0.099
nd
nd
1.063
2.1
a
b
Ki values were determined using multiple concentrations of [14C]SPD in 5 min initial rate transport assays. L1210 cells were cultured
for 24 h with the compounds; intracellular levels of the acridine conjugates were determined by fluorimetry; mean value ((15%) from
d
three determinations; nd, not determined. c L1210 cells were incubated with the conjugates in the presence of 500 µM SPD. Differential
accumulation between control and SPD-treated cells. e L1210 cells were pretreated for 24 h with 5 mM DFMO before conjugate addition.
Ta ble 4. Cellular Uptake of the Acridine Conjugates in CHO
and CHO-MG Cells
L1210 and CHO cells, (ii) the effect of an excess of free
SPD on the accumulation of conjugates in L1210 cells,
cellular uptake (nmol/mg protein)a
(iii) the relative growth inhibitory effect and accumula-
tion of the conjugates in CHO and polyamine transport
deficient CHO-MG cells, and (iv) the effect of DFMO
on the growth inhibitory effect and the uptake of the
conjugates.
concn
conjugate
(µM)
CHO
CHO-MG
CHO/CHO-MG
8
10
2.5
10
1.25
10
2
0.5
0.5
0.5
0.100
0.014
0.183
0.018
0.125
0.034
0.167
0.081
0.210
0.108
0.017
0.172
0.013
0.112
0.032
0.054
0.026
0.061
0.93
0.83
1.1
1.4
1.1
1.1
3.1
3.1
3.4
9
(i) All six acridine conjugates competitively inhibited
the transport of [14C]SPD in L1210 cells with Ki values
around 0.1 µM (Table 3). In both series, the lowest Ki
value was observed with the conjugates tethered with
four methylene units (9 and 12). Noteworthy, all deriva-
tives had Ki values approximatively 10 times lower than
SPM itself. The coupling of a hydrophobic heterocyclic
or aromatic moiety to SPM 1 may improve its affinity
for the polyamine transporter. Conjugates 10 and 11
also inhibited the uptake of [14C]SPD in CHO cells in a
dose-dependent manner with IC50 values of 37 and 60
nM, respectively (Figure 5). Altogether, these data
demonstrate that the spermine-acridine conjugates
have a high affinity for the PTS.
10
11
12
13
a
Conjugates were added 24 h after seeding; intracellular levels
of the conjugates were determined by fluorimetry in cells treated
with the conjugates during 24 h; mean value ((15%) from three
determinations.
on the secondary amino group: the N1 spermine conju-
gate 13 (IC50 ) 1.4 µM) is 16 times more active on L1210
cells than 6, its N4 homologue33 (IC50 ) 23 µM). All
conjugates were also more toxic than unsusbtituted
acridine (IC50 ) 45 µM),58 but 7, the nonpolyamine-
containing 9-(N-butyl)aminoacridine (IC50 ) 2 µM),33
was as potent as its spermine-containing homologue 12.
To check whether the differential toxicity of the two
families of acridine conjugates is due to their cellular
uptake, we measured the cellular conjugate concentra-
tions in L1210 and CHO cells after 24 h. Conjugates
8-13 were taken up by the cells in a dose-dependent
manner (Tables 3 and 4, Figure 4). The aminoacridines
11-13 were more readily taken up than the amido-
acridines 8-10. At concentrations of 10 µM, the intra-
cellular concentrations of 8-10 in L1210 cells were at
best equivalent to those measured in cells treated with
2 µM 11-13. Moreover, L1210 and CHO cells treated
with 9, the more potent of the three amidoacridines, had
significantly higher intracellular concentrations of the
conjugate. Thus, the differential activity of the two
families of acridine conjugates is at least related to their
differential accumulation in the cells.
(ii) Taking into account the affinity of the acridine
conjugates deduced from the Ki values (Table 3), 500
µM SPD would inhibit almost completely the uptake of
the conjugates via the PTS in L1210 cells. As shown in
Table 3, the presence of SPD in the culture medium
reduced only partially the amount of conjugates taken
up by the cells. In the range of concentrations studied,
an excess of SPD reduced cellular uptake by a similar
amount (around 0.015 and 0.100 nmol/mg for amido-
and aminoacridines, respectively). These amounts are
likely to reflect the actual quantity of conjugates that
entered the cells via the PTS. These data demonstrate
that the amount of conjugates transiting through the
PTS is relatively small and that the uptake rate through
the PTS is higher for the aminoacridines.
(iii) While conjugates 8-10 had similar growth in-
hibitory effects on CHO and CHO-MG, conjugates 11-
13 were 3-4 times more active on CHO than CHO-MG
cells (Table 2; Figure 3). These effects were correlated
with the amount of derivatives accumulated in the cells:
CHO and CHO-MG cells accumulated similar amounts
of amidoacridines (8-10) while intracellular concentra-
4. In ter a ction w ith th e P TS. To determine whether
the intracellular accumulation of the conjugates was
dependent on the PTS, we determined (i) the ability of
the conjugates to compete with [14C]SPD for uptake in

5104 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Delcros et al.
F igu r e 4. Cellular uptake of the conjugates 10 and 11 in control or DFMO-pretreated CHO and mutant polyamine transport
deficient CHO-MG cells. Cells pretreated or not for 24 h with 5 mM DFMO were exposed for 24 h to the conjugates. Intracellular
concentrations of the conjugates were determined by fluorimetry.
F igu r e 5. Effect of short term exposure to the conjugates 10 and 11 on the polyamine transport activity in control or DFMO-
pretreated CHO cells. CHO cells grown for 24 h with or without 5 mM DFMO were exposed for 2 h to various concentrations of
the conjugates. [14C]SPD uptake activity was measured after conjugate removal and washings. The [14C]SPD transport activities
in control and DFMO-pretreated cells were 0.29 ( 0.01 and 0.41 ( 0.03 nmol/mg/min, respectively.
tions of aminoacridines (11-13) were significantly
higher (approximately 3-fold) in CHO than in CHO-MG
cells (Table 4; Figure 4).
curves in the presence of DFMO are shown in Figure 3.
For the amidoacridines 8-10, the curves were biphasic
and showed a strong synergistic effect at low concentra-
tions (Figure 3). Consequently, DFMO decreased more
dramatically the IC25 than the IC50 values. As shown
in Table 2, in the presence of DFMO, IC50 values for
conjugates 8-10 were 6-19 times lower while their IC25
values were 100-240 times lower. The effect of DFMO
on the aminoacridines 11-13 was less dramatic (Figure
3; Table 2). DFMO reduced the IC50 values by a factor
of 2-3 and the IC25 values by a factor of 2-10 (Table
2). In contrast, DFMO had no effect on the toxicity of
the drugs on CHO-MG cells, which demonstrates that
the synergistic effect observed with DFMO is strictly
dependent on the presence of an active PTS.
Altogether, these results strongly suggest that the
uptake of the acridine conjugates is not solely dependent
on the polyamine transport apparatus. The contribution
of the PTS to the overall accumulation is more impor-
tant for aminoacridines than for their amide analogues
but still relatively poor. As a comparison, the sensitivity
of CHO cells to bis(7-amino-4-azaheptyl)dimethylsilane,
a polyamine analogue taken up by the polyamine
transporter, was higher by several orders of magnitude
than the mutant cell line CHO-MG.64 The architecture
of the derivatives influences both the polyamine trans-
porter-dependent and -independent uptake with a
marked preference for the aminoacridines.
(iv) Treatment of L1210, CHO, and CHO-MG cells
with DFMO, an inhibitor of ornithine decarboxylase,
results in a depletion of intracellular pools of putrescine
and SPD. This reduction of intracellular polyamine
levels induced an up-regulation of the activity of the
PTS in L1210 and CHO cells.65,66 Exposure of L1210 or
CHO cells to the acridine derivatives in the presence of
5mM DFMO enhanced their growth inhibitory activity.
Typical examples of dose-dependent growth inhibitory
Pretreatment of L1210 or CHO cells with 5 mM
DFMO for 24 h increased the uptake of the acridine
derivatives but was ineffective in CHO-MG cells (Table
3; Figure 4). Intracellular accumulation of the acridine
conjugates was enhanced 2-3 times in DFMO-pre-
treated L1210 cells when compared to controls. The
dose-dependent accumulation of the conjugates 10 and
11 in CHO and CHO-MG cells pretreated with DFMO
was studied over a large concentration range. While
DFMO had no effect on the accumulation of both

Effect of Spermine Conjugation on Acridine
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5105
conjugates in CHO-MG, their accumulation was im-
proved in CHO cells as observed in L1210 cells. A
remarkable increase was observed with the amido-
acridine 10 at concentrations below 0.5 µM, a concen-
tration for which accumulation of the conjugate is below
detection level in control cells.
inhibition rates were obtained when the conjugates were
applied in the presence of 200 µM cycloheximide (data
not shown). Following from the potent dose-dependent
inhibitory activity of the acridine-spermine conjugates,
observed when the conjugates were present in the assay
medium, we conclude that the acridine conjugates bind
very rapidly and avidly to the transporter. A slow rate
of dissociation of the conjugate from the transporter, as
hypothesized by Weeks et al.,70 would prevent the
recovery of a full transport activity after removal of the
conjugates. Another potential mechanism may involve
an interaction with calmodulin. Calmodulin antagonists
inhibit polyamine uptake,71-73 and conjugates of sper-
mine with lipophilic substituents, such as N1-dansyl-
spermine and N1-(n-octanesulfonyl)spermine, potent
calmodulin antagonists.62 The exact mechanism of the
inhibition of the PTS by the acridine conjugates remains
to be fully investigated. It appears obvious from these
observations that the repression of the activity of the
PTS induced by the acridine conjugates limits their own
accumulation via the polyamine transporter and ac-
counts for the low amounts of drugs transiting through
the PTS.
Altogether, from the DFMO studies, we conclude that
all these derivatives are able to use the PTS in addition
to another independent transport mechanism. The effect
of the derivatives on cell growth is dependent on their
intracellular concentration, and the effect of DFMO on
the cytotoxicity is tentatively explained as follows. For
the amidoacridines, the amount of drug transiting
through the PTS in normal cells is likely to be too low
to exert a cytotoxic effect and the observed toxicity is
solely due to the drug penetrating through a polyamine
transport-independent system. In contrast, in cells
treated with DFMO, the amount of drug taken up by
the activated system passes the threshold beyond which
a cytotoxic effect can be exerted. Because of the high
affinity of the drug for the PTS, these effects are
observed at low concentrations. However, for concentra-
tions over 0.1 µM, intracellular concentrations (as well
as the effect on cell growth) plateaued. The combined
effects of the drugs taken up by the two uptake mech-
anisms will give an overall dose-dependent biphasic
effect. The plateau may be the consequence either of a
saturation of the transport system or of a down-
regulation of the system preventing higher amounts of
drugs being taken up as discussed below. In contrast,
higher amounts of aminoacridines transit through the
PTS and the threshold is already reached in normal
cells. DFMO improves their effect on cell growth in
proportion to the amount of drug taken up by the
activated transport system.
Con clu sion s
Polyamine vectorization is believed to be a valu-
able strategy to increase the selectivity of anti-
cancer agents. However, little is known on the feasi-
bility of this approach in terms of drug delivery. The
acridine-spermine conjugates were designed as test
compounds to study the influence of polyamine conjuga-
tion on cytotoxicity and on the interactions with the
PTS.
Our data demonstrate that the concept of polyamine
vectorization will successfully depend on the relative
amount of drug carried by the specific transporter or
using other entry pathways. A selectivity will be reached
(i) if the affinity of the drug for the PTS is higher than
for other transport system(s) and (ii) if the amount of
drug carried by the specific transporter is sufficient to
pass the threshold required for the desired biological
activity. It is however expected that the tumor selectiv-
ity of drugs vectorized by polyamines will need the
cotreatment with DFMO to up-regulate the PTS activity
of the tumor cells. In this regard, it is important to note
that despite the fact that the spermine conjugates 11-
13 were as potent as their nonpolyamine-containing
homologue 7 in normal cells, DFMO has a synergistic
effect on the toxicity of conjugates 11-13, an effect
dependent on the presence of an active PTS. Such an
effect is not anticipated for conjugate 7 because it is
certainly not transported by the PTS.
However, the benefits expected from the vectorization
by spermine may be diminished by the ability of the
conjugates to repress the activity of the PTS of the
target cells and therefore to inhibit their own transport.
This effect limits the amount of drug transported
selectively into the cells. The elucidation of the mech-
anism of this transport inhibition is under investigation.
Our future studies will focus on the design of polyamine
conjugates devoid of this disadvantageous property.
Modifications of the spermine chain may be a promising
approach because in another series of polyamine con-
jugates, we observed that the substitution of spermine
Because of the high velocity of the PTS, this system
would allow a significant accumulation of spermine
conjugates in cells. However, the activity of the PTS is
highly regulated. In particular, the activity of the PTS
is repressed in response to elevated intracellular levels
of natural polyamines via the induction of antizyme.22,67
Feedback control of uptake can also be mimicked by
structural analogues of polyamines,68,69 which indicates
that analogues may inhibit their own uptake. In a
recent paper, Weeks et al.70 have shown that the
treatment of cells for a short period with a lysine-SPM
conjugate, a potent competitive inhibitor of polyamine
transport, results in a dose-dependent, antizyme-
independent repression of the polyamine transport
activity. An immediate increase in transport was ob-
served upon removal of the lysine-SPM conjugate, but
the recovery of a full transport activity required several
hours of culture in the absence of any polyamine
conjugates.70 When CHO cells were cultured for 2 h with
various concentrations of 10 and 11, the activity of the
PTS, measured in a 5 min assay after removal of the
conjugates, was inhibited in a dose-dependent manner
with IC50 values around 25 nM (Figure 5). This inhibi-
tory effect was independent of the initial activity of the
PTS as evidenced by the similar inhibition rates deter-
mined on DFMO-pretreated cells. These results dem-
onstrate that acridine-spermine conjugates induce a
repression of the transport activity in CHO cells. A role
for antizyme in this process is excluded because similar

5106 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Delcros et al.
128.10, 128.48 (CH-Ar), 136.37 (OCH2CC5H5), 157.29 (CdO).
MS-FAB (m/z) calcd for C11H16NO3 (M + H), 210.25; found,
210.20.
Da ta fr om N-Ben zyloxyca r bon yl-4-a m in obu ta n -1-ol
(15). Compound 15 was prepared from 4-aminobutan-1-ol and
benzylchloroformate; purified by recrystallization with AcOEt/
hexane; white solid; 90%; Rf 0.34. 1H NMR (270 MHz,
CDCl3): δ 1.57 (m, 4H, CH2CH2CH2CH2), 2.02 and 2.39 (br s,
1H, OH), 3.20 (m, 2H, CH2NH), 3.63 (m, 2H, CH2OH), 5.08 (s,
2H, OCH2C6H5), 7.26-7.34 (m, 5H, H-Ar). 13C NMR (68 MHz,
CDCl3): δ 26.41 and 29.55 (CH2CH2CH2CH2), 40.74 (CH2NH),
62.19 (CH2OH), 66.56 (OCH2C6H5), 128.02, 128.44 (CH-Ar),
136.54 (OCH2CC5H5), 156.55 (CdO). MS-FAB (m/z) calcd for
F igu r e 6. Example of atom numbering used for NMR assign-
ments.
with some synthetic homologous chain reduced the
ability of the conjugates to repress the activity of the
PTS without altering their affinity for the transporter
(unpublished data).
C
12H18NO3 (M + H), 224.27; found, 224.20.
Da ta fr om N-Ben zyloxyca r bon yl-5-a m in op en ta n -1-ol
(16). Compound 16 was prepared from 5-aminopentan-1-ol and
benzylchloroformate; purified by recrystallization with AcOEt/
hexane; white solid; 84%; Rf 0.43. 1H NMR (270 MHz,
CDCl3): δ 1.35-1.43 and 1.47-1.62 (m, 6H, CH2CH2CH2CH2-
CH2), 1.84 (br s, 1H, OH), 3.20 (m, 2H, CH2NH), 3.62 (t, 2H,
J ) 6.2 Hz, CH2OH,), 5.08 (s, 2H, OCH2C6H5), 7.26-7.36 (m,
5H, H-Ar). 13C NMR (68 MHz, CDCl3): δ 22.82, 26.67 and
32.14 (CH2CH2CH2CH2CH2), 40.87 (CH2NH), 62.52 (CH2OH),
66.56 (OCH2C6H5), 128.04, 128.46 (CH-Ar), 136.57 (OCH2-
CC5H5), 156.48 (CdO). MS-FAB (m/z) calcd for C13H20NO3
(M + H), 238.30; found, 238.20.
Gen er a l P r oced u r e of Sw er n Oxid a tion . Oxalyl chloride
(1.1 equiv) was dissolved in 15 mL of anhydrous CH2Cl2 and
cooled to -78 °C (ice dry and acetone). Anhydrous dimethyl
sulfoxide (DMSO, 2 equiv) was then added dropwise over 5
min using a syringe, and the reaction mixture was stirred at
-78 °C for 10 min. N-Benzyloxycarbonyl-amino alcohol (1
equiv) dissolved in CH2Cl2 (35 mL) was added dropwise over
5 min, and a yellow precipitate was formed. The reaction
mixture was then warmed until the precipitate dissolved. The
reaction mixture was cooled to -78 °C and stirred for 10 min.
Et3N (5 equiv) was then added, the reaction was warmed to
room temperature, and water (50 mL) was added. After the
organic layer was separated, the aqueous layer was extracted
with CH2Cl2 (2 × 50 mL), the combined organic layers were
dried with MgSO4, and then, the solvents were evaporated
under reduced pressure. The residue was purified by column
chromatography in AcOEt/hexane 40/60 and then 50/50. TLC
was performed in AcOEt/hexane 40/60 and visualized using
anisaldehyde.
Exp er im en ta l Section
Reagent-grade solvents were purchased from chemical
companies and used directly without further purification
unless otherwise specified. Dichloromethane (CH2Cl2) was
distilled over P2O5 under nitrogen. Methanol (MeOH) was
distilled over Mg and I2 prior to use. Elemental analyses were
performed by the Laboratoire de Microanalyses (Faculte´ de
Pharmacie, Universite´ Paris XI, Chatenay-Malabry, France).
Purifications by column chromatography were carried out over
70-230 mesh silica gel (Merck) using various eluants as
specified in the text. Thin-layer chromatography (TLC) was
routinely carried out with aluminum sheets precoated with
silica gel 60 F254 (layer thickness: 0.22 mm) (Merck). Solvent
systems (expressed in volume percents) and Rf are indicated
in the text. The compounds were visualized under UV light
and staining with KMnO4, anisaldehyde, or ninhydrin. Fourier
transform IR (FTIR) spectra were recorded on a Perkin-Elmer
377 instrument (KBr pellets, ν: cm-1). NMR spectra were
recorded on a Bruker DMX spectrometer at 500 MHz (1H) or
125 MHz (13C), on a J EOL EX400 spectrometer at 400 MHz
(1H) or 100 MHz (13C), or on a J EOL GX270 spectrometer at
270 MHz (1H) or 68 MHz (13C). Tetramethylsilane (TMS) was
used as the internal standard for NMR spectra recorded in
CDCl3. 3-(Trimethylsilyl)-1-propanesulfonic acid sodium salt
(DSS) was used as the external standard for NMR spectra
recorded in D2O. Broad band and gated decoupling 13C NMR
spectra were recorded, and the assignments were made using
chemical shifts and coupling constants. High-resolution mass
spectra (HRMS), determined by liquid secondary-ion mass
spectrometry (LSIMS), were recorded on a ZabSpec Tof Micro-
mass instrument (Centre Re´gional de Mesures Physiques de
l’Ouest, Rennes, France) with a source temperature of 140 °C,
an ion (Cs+) accelerating potential of 8 kV, and mNBA (meta-
nitrobenzyl alcohol) as the matrix. Low-resolution mass spec-
tra (LRMS) were determined by chemical ionization (MS-CI)
or by fast atomic bombardment (MS-FAB) recorded on a VG
autospec. As indicated in Figure 6, the compounds were
numbered for the heterocyclic moiety using the IUPAC rules
and for the polyamine moiety with letters a-o.
Da ta for N-Ben zyloxyca r bon yl-3-a m in op r op a n a l (17).
Compound 17 was prepared from 14; white solid; 50%; Rf
1
0.32. H NMR (270 MHz, CDCl3): δ 2.74 (t, 2H, J ) 5.8 Hz,
CH2CHO), 3.49 (m, 2H, CH2NH), 5.08 (s, 2H, OCH2C6H5),
7.26-7.34 (m, 5H, H-Ar), 9.79 (s, 1H, CHO). 13C NMR (100
MHz, CDCl3): δ 34.47 (CH2NH), 44.09 (CH2CHO), 66.77
(OCH2C6H5), 128.11, 128.18, 128.55 (CH-Ar), 136.37 (OCH2-
CC5H5), 156.31 (CdO), 201.22 (CHO). MS-FAB (m/z) calcd for
C
11H14NO3 (M + H), 208.23; found, 208.10.
Da ta for N-Ben zyloxyca r bon yl-2-h yd r oxyp yr r olid in e
1. Ch em ica l Syn th eses. Gen er a l P r oced u r e for Am in o
Alcoh ol Syn th esis u n d er Sch otten -Ba u m a n Con d ition s.
The amino alcohol (3-aminopropan-1-ol, 4-aminobutan-1-ol, or
5-aminopentan-1-ol, 2 g) was dissolved at 0 °C in aqueous
NaOH (1.1 equiv). Benzylchloroformate (1.1 equiv) was added
dropwise. The reaction mixture was allowed to warm to room
temperature and stirred overnight. After the solvents were
evaporated under reduced pressure, the residue was purified.
TLC was performed in 200/10/1 CH2Cl2/MeOH/NH4OH and
visualized using KMnO4.
Data for N-Ben zyloxycar bon yl-3-am in opr opan -1-ol (14).
Compound 14 was prepared from 3-aminopropan-1-ol and
benzylchloroformate; purified by column chromatography (200/
10/1 CH2Cl2/MeOH/NH4OH); white solid; 97%; Rf 0.30. 1H
NMR (270 MHz, CDCl3): δ 1.69 (m, 2H, CH2CH2CH2), 2.03
and 2.95 (br s, 1H, OH), 3.30-3.37 (m, 2H, CH2NH), 3.66 (m,
2H, CH2OH), 5.09 (s, 2H, OCH2C6H5), 7.30-7.34 (m, 5H,
H-Ar). 13C NMR (68 MHz, CDCl3): δ 32.43 (CH2CH2CH2),
37.69 (CH2NH), 59.47 (CH2OH), 66.78 (OCH2C6H5), 128.02,
(18). Compound 18 was prepared from 15; yellow solid; 87%;
Rf 0.34. 1H NMR (270 MHz, CDCl3, mixture of conformational
isomers): δ 1.86-2.04 (m, 4H, NCH2CH2CH2CHOH), 3.13 and
3.95 (br s, 1H, OH), 3.36 (m, 1H, NCH2), 3.58 (m, 1H, NCH2),
5.17 (s, 2H, OCH2C6H5), 5.48-5.54 (m, 1H, OHCHN), 7.26-
7.37 (m, 5H, H-Ar). 13C NMR (100 MHz, CDCl3, mixture of
conformational isomers): δ 22.05 and 22.79 (NCH2CH2CH2-
CHOH), 32.66 and 33.58 (NCH2CH2CH2CHOH), 45.78 and
46.23 (NCH2), 66.90 and 67.14 (OCH2C6H5), 81.21 and 82.00
(OHCHN), 127.87, 128.09, 128.29, 128.53 and 128.66 (CH-
Ar), 136.37 and 136.47 (OCH2CC5H5), 154.15 and 155.50
74
(CdO). MS-CI (m/z) calcd for C12H16NO3 (M + H), 222.26;
found, 222.10.
Da t a for N-Ben zyloxyca r bon yl-2-h yd r oxyp ip er id in e
(19). Compound 19 was prepared from 16; white solid; 73%;
Rf 0.47. 1H NMR (270 MHz, CDCl3, mixture of conformational
isomers): δ 1.81-2.10 (m, 6H, NCH2CH2CH2CH2CHOH), 3.07
and 3.95 (br s, 1H, OH), 3.35 (m, 1H, NCH2), 3.59 (m, 1H,
NCH2), 5.14 (s, 2H, OCH2C6H5), 5.49-5.54 (m, 1H, OHCHN),

Effect of Spermine Conjugation on Acridine
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5107
7.27-7.37 (m, 5H, H-Ar). 13C NMR (68 MHz, CDCl3, mixture
of conformational isomers): δ 21.99 and 22.72 (NCH2CH2CH2-
CH2CHOH), 32.62 and 33.54 (NCH2CH2CH2CH2CHOH), 45.71
and 46.17 (NCH2), 66.85 and 67.08 (OCH2C6H5), 81.14 and
81.91 (OHCHN), 127.81, 128.02, 128.22, 128.46 and 128.59
(CH-Ar), 136.31 and 136.42 (OCH2CC5H5), 153.51 and 154.35
(CdO). MS-CI (m/z) calcd for C13H18NO3 (M + H), 236.29;
found, 236.10.
P r oced u r e for N1,N4,N9-Tr i-ter t-bu toxyca r bon ylsp er -
m in e Syn th esis (20). Compound 20 was made as previously
described;49 44%; Rf 0.38 (CH2Cl2/MeOH/NH4OH 70/10/1 and
visualized using ninhydrin). 1H NMR (400 MHz, CDCl3): δ
1.28-1.48 (m, 31H, 3(CH3)3C, H-6, H-7), 1.59-1.66 (m, 4H,
H-2, H-11), 2.74 (m, 2H, H-12), 3.00-3.45 (m, 12H, H-1, H-3,
H-5, H-8, H-10, NH2). 13C NMR (100 MHz, CDCl3): δ 25.42
and 26.02 (C-6, C-7), 28.09 and 28.93 (3(CH3)3C, C-2), 30.15
and 32.30 (C-11), 37.36 and 37.71 (C-1), 38.35 and 39.35 (C-
12), 43.54 and 44.42 (C-3, C-10), 46.43 and 46.82 (C-5, C-8),
78.96 and 79.74 ((CH3)3C), 155.52 and 156.21 (CdO).
2H, H-17), 3.16-3.64 (m, 14H, H-2, H-4, H-6, H-9, H-11, H-13,
H-15). MS-FAB (m/z) calcd for C33H66N5O8 (M + H), 660.91;
found, 660.30.
Da t a for N1,N5,N10,N14-Tet r a -ter t-b u t oxyca r b on yl-
1,5,10,14,19-p en ta a za n on a d eca n e (25). Compound 25 was
prepared from 22; colorless oil; 32%; Rf, 0.10 (c). 1H NMR (500
MHz, CDCl3): δ 1.25-1.88 (m, 48H, 4(CH3)3C, H-3, H-7, H-8,
H-12, H-16, H-17), 2.17 (br s, 2H, NH2), 2.90 (m, 2H, H-18),
3.10-3.42 (m, 14H, H-2, H-4, H-6, H-9, H-11, H-13, H-15). MS-
FAB (m/z) calcd for C34H68N5O8 (M + H), 674.94; found, 674.30.
Da t a for N1,N5,N10,N14-Tet r a -ter t-b u t oxyca r b on yl-
1,5,10,14,20-p en ta a za eicosa n e (26). Compound 26 was pre-
1
pared from 23; colorless oil; 34%; Rf 0.10. H NMR (500 MHz,
CDCl3): δ 1.24-1.85 (m, 50H, 4(CH3)3C, H-3, H-7, H-8, H-12,
H-16, H-17, H-18), 3.02-3.35 (m, 16H, H-2, H-4, H-6, H-9,
H-11, H-13, H-15, H-19), 8.13 (br s, 2H, NH2). MS-FAB (m/z)
calcd for C35H70N5O8 (M + H), 688.96; found, 688.30.
Gen er a l P r oced u r e of Acyla tion . To a solution of 24-
26 (1 equiv) in DMF (3 mL) was added acridine-9-carboxylic
acid (1 equiv). After it was dissolved, N,N′-dicyclohexylcarbo-
diimide (1.2 equiv) and then N1-hydroxybenzotriazole (cata-
lytical amount, 0.01 equiv) were added. The reaction mixture
was stirred at room temperature for 48 h. The precipitate was
then removed by filtration. The solvents were evaporated
under reduced pressure, and the residue was purified by
column chromatography in MeOH/NH4OH 95/5. TLC was
performed in CH2Cl2/MeOH 95/5 and visualized using nin-
hydrin (a). The removal of Boc groups was achieved overnight
using a 0.9 M solution of HCl gas in AcOEt. TLC was
performed in isopropylamine/MeOH/CHCl3 2/4/4 and visual-
ized using ninhydrin (b).
Da t a for N1-(Acr id in -9-ylca r b on yl)-1,5,9,14,18-p en t a -
a za octa d eca n e P en ta h yd r och lor id e (8). Boc-protected 27
was prepared from 24 (yellow oil; 64%; Rf 0.26 (a)). Deprotec-
tion gave 8 in 93% yield. Yellow solid; Rf 0.06 (b). FTIR 2300-
3700 (NH+), 1636 (CdN + CdO). 1H NMR (500 MHz, D2O):
δ 1.79-1.83 (m, 4H, H-h, H-i), 2.11 (m, 2H, H-b), 2.15-2.29
(m, 4H, H-e, H-l), 3.10-3.37 (m, 14H, H-c, H-d, H-f, H-g, H-j,
H-k, H-m), 3.83 (t, 2H, J ) 7 Hz, H-a), 7.97 (t, 2H, J ) 7 Hz,
H-3, 6), 8.27-8.31 (m, 4H, H-2, 7 and H-4, 5), 8.38 (d, 2H, J )
9 Hz, H-1, 8). 13C NMR (125 MHz, D2O): δ 23.11, 23.16 (C-e,
C-h, C-i), 24.13 (C-l), 25.88 (C-b), 36.93 (C-m), 37.83 (C-a),
44.88, 44.93, 45.03 (C-d, C-f, C-k), 46.01 (C-c), 47.23 and 47.39
(C-g, C-j), 120.62 (C-1, 8), 122.70 (C-12, 13), 126.47 (C-4, 5),
129.73 (C-3, 6), 138.07 (C-2, 7), 140.57 (C-11, 14), 150.32 (C-
9), 166.83 (CdO). HRMS (LSIMS) (m/z) calcd for C27H41N6O
(M + H), 465.3342; found, 465.3347. Anal. (C27H40N6O‚5HCl‚
3H2O) calcd: C, 46.44; H, 7.36; N, 12.04. Found: C, 45.96; H,
7.63; N, 12.06.
Da ta for N1-(Acr id in -9-ylca r bon yl)-1,6,10,15,19-p en ta -
a za n on a d eca n e P en ta h yd r och lor id e (9). Boc-protected 28
was prepared from 25 (yellow oil; 54%; Rf 0.23 (a)). Deprotec-
tion gave 9 in 96% yield. Yellow solid; Rf 0.07 (b). FTIR 2300-
3700 (NH+), 1638 (CdN + CdO). 1H NMR (500 MHz, D2O):
δ 1.80-1.85 (m, 4H, H-i, H-j), 1.91-1.96 (m, 4H, H-b, H-c),
2.10-2.20 (m, 4H, H-f, H-m), 3.10-3.23 (m, 14H, H-d, H-e,
H-g, H-h, H-k, H-l, H-n), 3.78 (m, 2H, H-a), 7.97 (t, 2H, J ) 7
Hz, H-3, 6), 8.27-8.30 (m, 4H, H-2, 7 and H-4, 5), 8.38 (d, 2H,
J ) 9 Hz, H-1, 8). 13C NMR (125 MHz, D2O): δ 23.10, 23.16
(C-f, C-i, C-j), 23.79 (C-c), 24.13 (C-m), 25.94 (C-b), 36.94 (C-
n), 40.18 (C-a), 44.86, 44.90, 44.93 (C-e, C-g, C-l), 47.40 and
47.42 (C-h, C-k), 47.78 (C-d), 120.32 (C-1, 8), 122.65 (C-12, 13),
126.54 (C-4, 5), 129.76 (C-3, 6), 138.26 (C-2, 7), 140.26 (C-11,
14), 150.91 (C-9), 166.41 (CdO). HRMS (LSIMS) (m/z) calcd
for C28H43N6O (M + H), 479.3498; found, 479.3507. Anal.
(C28H42N6O‚5HCl‚H2O) calcd: C, 49.73; H, 7.30; N, 12.43.
Found: C, 50.02; H, 7.57; N, 12.34.
Da ta for N1-(Acr id in -9-ylca r bon yl)-1,7,11,16,20-p en ta -
a za eicosa n e P en t a h yd r och lor id e (10). Boc-protected 29
was prepared from 26 (yellow oil; 67%; Rf 0.26 (a)). Deprotec-
tion gave 10 in 84% yield. Yellow solid; Rf 0.10 (b). FTIR 2300-
3700 (NH+), 1638 (CdN + CdO). 1H NMR (500 MHz, D2O):
δ 1.59 (m, 2H, H-c), 1.82-1.89 (m, 8H, H-b, H-d, H-j, H-k),
2.10-2.20 (m, 4H, H-g, H-n), 3.12-3.22 (m, 14H, H-e, H-f, H-h,
Gen er a l Meth od of Red u ctive Am in a tion . Compound
17, 18, or 19 (1 equiv) was dissolved with 20 (1.4 equiv) in
anhydrous MeOH (10 mL), and the solution was stirred on
molecular sieves 4 Å under N2 at 25 °C. Sodium cyanoboro-
hydride (1.7 equiv) and a catalytic amount of glacial acetic acid
(0.1 mL) were then added, and the reaction mixture was
stirred at 25 °C for 24 (for 17), 48 (for 18), or 72 h (for 19).
The mixture was then filtered and concentrated under reduced
pressure. The residue was then chromatographed in CH2Cl2/
MeOH/NH4OH 100/10/1. TLC was performed in CH2Cl2/
MeOH/NH4OH 100/10/1 and visualized using ninhydrin.
Da ta for N1,N5,N10-Tr i-ter t-bu toxyca r bon yl-N18-ben zyl-
oxyca r bon yl-1,5,10,14,18-p en ta a za octa d eca n e (21). Com-
1
pound 21 was prepared from 17; yellow oil; 54%; Rf 0.34. H
NMR (400 MHz, CDCl3): δ 1.44-1.45 (m, 31H, 3(CH3)3C, H-7,
H-8), 1.69 (m, 6H, H-3, H-12, H-16), 2.07 (br s, 1H, NH), 2.56-
2.69 (m, 4H, H-13, H-15), 3.13-3.48 (m, 12H, H-2, H-4, H-6,
H-9, H-11, H-17), 5.09 (s, 2H, OCH2C6H5), 7.30-7.36 (m, 5H,
H-Ar). MS-FAB (m/z) calcd for C36H64N5O8 (M + H), 694.93;
found, 694.30.
Da ta for N1,N5,N10-Tr i-ter t-bu toxyca r bon yl-N19-ben zyl-
oxyca r bon yl-1,5,10,14,19-p en ta a za n on a d eca n e (22). Com-
pound 22 was prepared from 18; colorless oil; 40%; Rf 0.36.
1H NMR (500 MHz, CDCl3): δ 1.44-2.00 (m, 40H, 3(CH3)3C,
H-3, H-7, H-8, H-12, H-16, H-17, NH), 2.56-2.89 (m, 4H, H-13,
H-15), 3.10-3.33 (m, 12H, H-2, H-4, H-6, H-9, H-11, H-18),
5.09 (s, 2H, OCH2C6H5), 7.30-7.33 (m, 5H, H-Ar). MS-FAB
(m/z) calcd for C37H66N5O8 (M + H), 708.95; found, 708.30.
Da ta for N1,N5,N10-Tr i-ter t-bu toxyca r bon yl-N20-ben zyl-
oxyca r b on yl-1,5,10,14,20-p en t a a za eicosa n e (23). Com-
1
pound 23 was prepared from 19; yellow oil; 29%; Rf 0.16. H
NMR (500 MHz, CDCl3): δ 1.23-2.06 (m, 42H, 3(CH3)3C, H-3,
H-7, H-8, H-12, H-16, H-17, H-18, NH), 2.78-2.92 (m, 4H,
H-13, H-15), 3.10-3.58 (m, 12H, H-2, H-4, H-6, H-9, H-11,
H-19), 5.09 (s, 2H, OCH2C6H5), 7.30-7.36 (m, 5H, H-Ar).
Gen er a l P r oced u r e of Tetr a Boc P en ta m in e F or m a -
tion . To a solution of 21, 22, or 23 (1 equiv) in dimethylform-
amide (DMF, 5 mL), di-tert-butyl dicarbonate (1.2 equiv)
dissolved in 1 mL of DMF was added at 0 °C under N2. The
reaction mixture was stirred at room temperature for 1 h.
Concentrated NH4OH (1 mL) was added, and the reaction
mixture was stirred for 20 min. The solvents were evaporated
under reduced pressure at 40 °C. Pearlman’s catalyst (1 equiv)
was then added to the residue dissolved in 3 mL of MeOH.
The mixture was stirred for 4 h under an atmosphere of H2 at
25 °C. The mixture was filtered, washed with MeOH, and
concentrated under reduced pressure. The residue was purified
by column chromatography in CH2Cl2/MeOH/NH4OH 100/10/
1. TLC was performed in CH2Cl2/MeOH/NH4OH 100/10/1 and
visualized using ninhydrin.
Da t a for N1,N5,N10,N14-Tet r a -ter t-b u t oxyca r b on yl-
1,5,10,14,18-p en ta a za octa d eca n e (24). Compound 24 was
prepared from 21; colorless oil; 63%; Rf 0.10. 1H NMR (400
MHz, CDCl3): 1.44-1.45 (m, 40H, 4(CH3)3C, H-7, H-8), 1.66-
1.75 (m, 6H, H-3, H-12, H-16), 1.99 (br s, 2H, NH2), 2.85 (m,

5108 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Delcros et al.
H-i, H-l, H-m, H-o), 3.72 (t, 2H, J ) 7 Hz, H-a), 7.89 (m, 2H,
H-3, 6), 8.16-8.21 (m, 6H, H-1, 8, H-2, 7 and H-4, 5). 13C NMR
(125 MHz, D2O): δ 23.11, 23.16 (C-g, C-j, C-k), 23.81 (C-c),
24.13 (C-n), 25.61 (C-d), 28.23 (C-b), 36.95 (C-o), 40.48 (C-a),
44.79, 44.91, 44.94 (C-f, C-h, C-m), 48.07 (C-e), 47.40 and 47.42
(C-i, C-l), 121.08 (C-1, 8), 122.47 (C-12, 13), 126.31 (C-4, 5),
129.52 (C-3, 6), 137.48 (C-2, 7), 140.94 (C-11, 14), 149.88 (C-
9), 166.57 (CdO). HRMS (LSIMS) (m/z) calcd for C29H45N6O
(M + H), 493.3655; found, 493.3659. Anal. (C29H44N6O‚5HCl‚
3H2O) calcd: C, 47.96; H, 7.63; N, 11.57. Found: C, 48.23; H,
7.71; N, 11.41.
Gen er a l P r oced u r e of Alk yla tion . Compound 24, 25, or
26 (1 equiv) and 9-phenoxyacridine (1 equiv, prepared as
previously described)50-52 was stirred with 5 g of phenol at 80
°C under a nitrogen atmosphere for 5 h. To the resulting
mixture were added CH2Cl2 (30 mL) and NaOH 1 N (20 mL).
After it was decanted, washed with NaOH 1 N (4 × 20 mL),
and dried over K2CO3, the organic layer was evaporated under
reduced pressure and then the residue was purified by column
chromatography with a mixture of CH2Cl2/MeOH/NH4OH 98/
2/0.2 and then 97/3/0.3, 96/4/0.4 and then 95/5/0.5. TLC was
performed in CH2Cl2/MeOH/NH4OH 95/5/0.5 and visualized
using ninhydrin (c). The removal of Boc groups was achieved
using a 0.9 M solution of HCl gas in ethyl acetate (16 h). TLC
was performed in isopropylamine/MeOH/CHCl3 2/4/4 and
visualized using ninhydrin (b).
Da t a for N1-(Acr id in -9-yl)-1,5,9,14,18-p en t a a za oct a -
d eca n e Hexa h yd r och lor id e (11). Boc-protected 30 was
prepared from 24 (yellow oil; 65%; Rf 0.36 (c)). Deprotection
gave 11 in 75% yield. Yellow solid; Rf 0.10 (b), 0.15 (MeOH/
NH4OH 70/30), 0.35 (MeOH/NH4OH 50/50). FTIR 2300-3700
(NH+), 1634 (CdN). 1H NMR (500 MHz, D2O): δ 1.79-1.85
(m, 4H, H-h, H-i), 2.09-2.20 (m, 4H, H-e, H-l), 2.26 (m, 2H,
H-b), 3.12-3.16 (m, 6H, H-g, H-j, H-m), 3.18-3.22 (m, 8H, H-c,
H-d, H-f, H-k), 3.93 (t, 2H, J ) 7.5 Hz, H-a), 7.31 (d, 2H, J )
9 Hz, H-1, 8), 7.41 (t, 2H, J ) 8 Hz, H-3, 6), 7.78 (t, 2H, J )
7.5 Hz, H-2, 7), 7.96 (d, 2H, J ) 9 Hz, H-4, 5). 13C NMR (125
MHz, D2O): δ 23.12, 23.16 (C-e, C-h, C-i), 24.14 (C-l), 26.27
(C-b), 36.93 (C-m), 44.86, 44.94, 45.10 (C-d, C-f, C-k), 45.40
(C-c), 45.89 (C-a), 47.23 and 47.40 (C-g, C-j), 111.78 (C-12, 13),
118.40 (C-1, 8), 124.45 (C-3, 6, C-4, 5), 135.66 (C-2, 7), 138.88
(C-11, 14), 156.96 (C-9). HRMS (LSIMS) (m/z) calcd for
465.3692. Anal. (C28H44N6‚6HCl) calcd: C, 49.44; H, 7.37; N,
12.35. Found: C, 49.13; H, 7.52; N, 12.07.
2. Biologica l Stu d ies. Unless otherwise stated, 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). Data are
given as mean values of three or more experiments. Compari-
sons between means were made using the Student’s t-test
assuming significance at p < 0.01.
a . Visible Absor p tion a n d F lu or im etr y. Visible absorp-
tion spectra were recorded on a Varian DMS 300 spectro-
photometer (Les Ulis, France), and the fluorescence excitation
and emission spectra were recorded on a Fluorolog 2 spectro-
fluorimeter (J obin Yvon, Lonjumeau, France) on conjugate
solution in 10 mM NaCl, 50 mM Tris-HCl (pH 7.4) or 0.2 N
HClO4 in the presence or absence of CT-DNA.
b. F lu or escen ce Bin d in g Stu d ies. Quenching Q values
were determined for CT-DNA, [poly(dA-dT)]2, and [poly(dG-
dC)]2 (Amersham Pharmacia Biotech, Saclay, France) for
solutions of 20 µM DNAp in acetate buffer (9.3 mM NaCl, 2
mM NaOAc, 0.1 mM ethylenediaminetetraacetic acid (EDTA),
pH 5.0) containing 2 µM ethidium bromide as described
previously.60 The CC50 values for ethidium displacement were
determined using solutions containing 1 µM CT-DNA and 1.26
µM ethidium bromide.58 Fluorescence (excitation at 546 nm;
emission at 595 nm) was monitored in 10 mm path length
quartz cuvettes using a Fluorolog 2 spectrofluorimeter follow-
ing serial addition of aliquots of a stock conjugate solution.
The Q and CC50 values are defined as the conjugate concentra-
tions reducing the fluorescence of initially DNA-bound ethid-
ium by 50%. None of the compounds showed fluorescence at
595 nm or interfered with the fluorescence of unbound
ethidium. Molar extinction values of ꢀ260 ) 6600, 6700, and
8400 (M phosphate)-1 cm-1, respectively, were used for [poly(dA-
dT)]2, CT-DNA, and [poly(dG-dC)]2.30,75
c. Cell Cu ltu r e. Murine leukemia L1210, CHO, and CHO-
MG cells were grown in RPMI 1640 medium (Eurobio, Les
Ulis, France) supplemented with 10% fetal calf serum, 2 mM
glutamine, penicillin (100 U/mL), and streptomycin (50 µg/
mL) (BioMerieux, Marcy l’Etoile, France). l-Proline (2 µg/mL)
was added to the culture medium for CHO-MG cells. Cells
were grown at 37 °C under a humidified 5% CO2 atmosphere.
As all of the conjugates were substrates of the bovine serum
amine oxidase (data not shown), 2 mM aminoguanidine was
added to the culture medium to prevent any oxidative degra-
dation of the conjugates by the enzyme present in the calf
serum.
C
26H41N6 (M + H), 437.3393; found, 437.3400.
Da ta for N1-(Acr id in -9-yl)-1,6,10,15,19-p en ta a za n on a -
d eca n e Hexa h yd r och lor id e (12). Boc-protected 31 was
prepared from 25 (yellow oil; 54%; Rf 0.33 (c)). Deprotection
gave 12 in 71% yield. Yellow solid; Rf 0.07 (b). FTIR 2300-
1
3700 (NH+), 1632 (CdN). H NMR (500 MHz, D2O): δ 1.80-
d . SP D Up ta k e In h ibition in L1210 a n d CHO Cells. The
acridine derivatives were studied for their ability to compete
with [14C]SPD (Amersham Pharmacia Biotech) for uptake into
L1210 cells in vitro. Cell suspensions (1.5 × 106 cells) were
incubated in 0.6 mL of Hanks’ balanced salt solution (HBSS)
supplemented with 20 mM Hepes containing 1, 2, and 4 µM
[14C]SPD alone or with the additional presence of 0.025, 0.050,
and 0.125 µM acridine derivative for 5 min at 37 °C. At the
end of the incubation period, the cell suspensions were layered
on top of a mixture of corn oil and dibutylphthalate and
centrifuged.62 Cell pellets were dissolved in 500 µL of 0.1 N
NaOH. Aliquots were transferred to vials for scintillation
counting. Other aliquots were used for protein determination.
Lineweaver-Burke plots indicated a simple competitive in-
hibition with respect to SPD.
1.84 (m, 6H, H-c, H-i, H-j), 1.92 (m, 2H, H-b), 2.11-2.17 (m,
4H, H-f, H-m), 3.12-3.20 (m, 14H, H-d, H-e, H-g, H-h, H-k,
H-l, H-n), 3.91 (t, 2H, J ) 7 Hz, H-a), 7.41 (d, 2H, J ) 9 Hz,
H-1, 8), 7.45 (t, 2H, J ) 7.5 Hz, H-3, 6), 7.84 (t, 2H, J ) 8 Hz,
H-2, 7), 8.04 (d, 2H, J ) 9 Hz, H-4, 5). 13C NMR (125 MHz,
D2O): δ 23.10, 23.15 (C-f, C-i, C-j), 23.38 (C-c), 24.13 (C-m),
26.48 (C-b), 36.93 (C-n), 44.87, 44.93 (C-e, C-g, C-l), 47.67 (C-
d), 48.39 (C-a), 47.39 and 47.42 (C-h, C-k), 118.41 (C-1, 8),
124.20 (C-3, 6, C-4, 5), 135.54 (C-2, 7), 157.37 (C-9). HRMS
(LSIMS) (m/z) calcd for C27H43N6 (M + H), 451.3549; found,
451.3542.
Data for N1-(Acr idin -9-yl)-1,7,11,16,20-pen taazaeicosan e
H exa h yd r och lor id e (13). Boc-protected 32 was prepared
from 26 (yellow oil; 59%; Rf 0.32 (c)). Deprotection gave 13 in
82% yield. Yellow solid; Rf 0.08 (b). FTIR 2300-3700 (NH+),
1
CHO cells were grown in 12 well plates. After three
washings in prewarmed phosphate-buffered saline (PBS), 1
mL of a prewarmed solution of HBSS-Hepes buffer containing
1 µM [14C]SPD was added to each well. When required, the
solution contained various concentrations of the conjugates.
After it was incubated at 37 °C for 5 min, the cells were washed
three times with ice-cold PBS and lysed in 0.1 N NaOH. In
some experiments, CHO cells were pretreated with 5 mM
DFMO during 24 h and/or incubated for 2 h at 37 °C with
various concentrations of conjugates (0.001-10 µM) in the
culture medium before the uptake measurements.
1632 (CdN). H NMR (500 MHz, D2O): δ 1.48 (m, 2H, H-c),
1.73-1.88 (m, 8H, H-b, H-d, H-j, H-k), 2.09-2.17 (m, 4H, H-g,
H-n), 3.08-3.20 (m, 14H, H-e, H-f, H-h, H-i, H-l, H-m, H-o),
3.82 (t, 2H, J ) 7.5 Hz, H-a), 7.37 (d, 2H, J ) 9 Hz, H-1, 8),
7.41 (t, 2H, J ) 8 Hz, H-3, 6), 7.80 (t, 2H, J ) 7.5 Hz, H-2, 7),
7.99 (d, 2H, J ) 9 Hz, H-4, 5). 13C NMR (125 MHz, D2O): δ
23.10, 23.15 (C-g, C-j, C-k), 23.51 (C-c), 24.13 (C-n), 25.67 (C-
d), 28.93 (C-b), 36.95 (C-o), 44.81, 44.89, 44.93 (C-f, C-h, C-m),
48.02 (C-e), 48.79 (C-a), 47.39 et 47.43 (C-i, C-l), 118.34 (C-1,
8), 124.12 (C-3, 6, C-4, 5), 135.48 (C-2, 7), 157.11 (C-9). HRMS
(LSIMS) (m/z) calcd for C28H45N6 (M + H), 465.37057; found,

Effect of Spermine Conjugation on Acridine
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e. In Vitr o Eva lu a tion of Con ju ga te Cytotoxicity/
Cytosta sy. The effects of the acridine derivatives on cell
growth were assayed in sterile 96 well microtiter plates
(Becton Dickinson, Oxnard, CA). L1210 cells were seeded at
5 × 104 cells/mL of medium (100 µL per well). Single CHO
and CHO-MG cells, harvested by trypsinization, were plated
at 2 × 103 cells/mL. Conjugate solutions (5 µL per well) of
appropriate concentration were added at the time of seeding
for L1210 cells and after an overnight incubation for CHO and
CHO-MG cells. In some experiments, 5 mM DFMO was added
to the culture medium at the time of conjugate addition. After
exposure to the conjugate for 48 h, cell growth was determined
by measuring formazan formation from 3-(4,5-dimethylthiazol-
2-yl)2,5-diphenyltetrazolium using a Titertek Multiskan MCC/
340 microplate reader (Labsystems, Cergy-Pontoise, France)
for absorbance (540 nm) measurements.76
f. Cellu la r Up ta k e. For determination of the cellular
uptake of the derivatives, cells were seeded in culture flasks
at 2 × 105 cells/mL for L1210 cells and at 1 × 105 cells/mL for
CHO and CHO-MG cells. Conjugates were added at the time
of seeding for L1210 and 24 h after seeding for CHO and CHO-
MG cells. In some experiments, 500 µM SPD was added at
the time of conjugate addition.
For cellular uptake in DFMO-pretreated L1210 cells, cells
were seeded at 105 cells/mL in culture medium containing
5mM DFMO and conjugates were added 24 h later. For cellular
uptake in DFMO-pretreated CHO and CHO-MG cells, cells
were seeded at 5 × 104 cells/mL. DFMO (5 mM) was added 24
h later. After they were incubated for 48 h, conjugates were
added as dilute solutions in 0.14 M NaCl.
All cells were harvested 24 h after conjugate addition.
Harvested cells were washed three times in 0.14 M NaCl. Cell
pellets were disrupted by sonication in 1 mL of 0.2 N HClO4/2
M NaCl. After 16 h at 4 °C, homogenates were centrifuged at
15 000 rpm for 30 min. Pellets were dissolved in 0.1 N NaOH
and used for protein determination with the Folin phenol
reagent.77 Conjugate concentrations in the supernatants were
evaluated by fluorescence measurements using dilutions of the
compounds in extraction buffer as standards. Fluorescence
spectra were recorded on a Fluorolog 2 spectrofluorimeter:
aminoacridine (exc: 360 nm; em: 380-500 nm); amidoacridine
(exc: 410 nm; em: 420-500 nm).
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Ack n ow led gm en t. We are indebted to Prof. W.
Flintoff (University of Western Ontario, London, Canada)
for providing the CHO-MG cells; to A. Grastien, S.
Duhieu, M. Foucault, and M. Le Roch for technical
assistance; and to Dr. F. Gaboriau for helpful discus-
sions. We acknowledge the Centre National de la
Recherche Scientifique (CNRS), the Association pour la
Recherche sur le Cancer (ARC), and the Ligue Nationale
Contre le Cancer (LNCCscommite´s d’Ille-et-Vilaine, du
Morbihan et des Coˆtes d’Armor) for financial support
and for fellowships to S.T. and to B.M. (LNCC). A
significant part of these studies was enabled by a grant
from the GP2A, The Atlantic Arc, for international
student exchange (S.T. and S.C.) between our labora-
tories, and we gratefully acknowledge this award. Our
sincere thanks are also due to A. Bondon (UMR 6509
CNRS, Rennes, France) for performing HMBC and
HMQC spectra and Dr. P. Guenot (Centre Re´gional de
Mesures Physiques de l’Ouest, Rennes, France) for mass
measurements.
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