Effect of polyamine homologation on the transport and biological properties of heterocyclic amidines

Article abstract of DOI:10.1021/jm050018q

Five sets of heterocyclic derivatives of various sizes and complexities coupled by an amidine function to putrescine, spermidine, or spermine were prepared. They were essentially tested to determine the influence of the polyamine chain on their cellular t

Full text of DOI:10.1021/jm050018q

232
J. Med. Chem. 2006, 49, 232-245
Effect of Polyamine Homologation on the Transport and Biological Properties of Heterocyclic
Amidines
Jean-Guy Delcros,*,†,‡ Sophie Tomasi,§ Ste´phane Duhieu,† Marine Foucault,† Be´ne´dicte Martin,†,‡ Myriam Le Roch,§
Vera Eifler-Lima,§,| Jacques Renault,§ and Philippe Uriac§
Groupe de Recherche en The´rapeutique Anticance´reuse, Faculte´ de Me´decine, Groupe Cycle Cellulaire, CNRS UMR 6061 “Ge´ne´tique et
De´Veloppement”, IFR 140 Ge´ne´tique Fonctionnelle Agronomie et Sante´, Faculte´ de Me´decine, et Substances liche´niques et photoprotection,
EA, Institut de Chimie de Rennes, Faculte´ de Pharmacie, UniVersite´ Rennes 1, 2 AVenue du Professeur Le´on Bernard, 35043 Rennes Cedex,
France, and Laborato´rio de Sintese de Fa´rmacos, Faculdade de Farma´cia, UniVersidade Federal do Rio Grande do Sul, AV. Ipiranga 2752,
Porto Alegre/RS, CEP 90610-000, Brazil
ReceiVed January 8, 2005
Five sets of heterocyclic derivatives of various sizes and complexities coupled by an amidine function to
putrescine, spermidine, or spermine were prepared. They were essentially tested to determine the influence
of the polyamine chain on their cellular transport. To comment on affinity and on selective transport via the
polyamine transport system (PTS), Ki values for polyamine uptake were determined in L1210 cells, and the
cytotoxicity and accumulation of the conjugates were determined in CHO and polyamine transport-deficient
mutant CHO-MG cells, as well as in L1210 and R-difluoromethylornithine- (DFMO-) treated L1210 cells.
Unlike spermine, putrescine and spermidine were clearly identified as selective motifs that enable cellular
entry via the PTS. However, this property was clearly limited by the size of substituents: these polyamines
were able to ferry a dihydroquinoline system via the PTS but did not impart any selectivity to bulkier
substituents.
Introduction
chlorambucil moiety to spermidine improved its in vitro
cytotoxicity when the nitrogen mustard functionality was bound
to the secondary amine; in contrast, no improvement resulted
from the linkage of chlorambucil to the C-5 of spermidine.24
However, the only spermidine conjugate tested in vivo was only
4-fold more potent than chlorambucil and did not show any
improvement of its therapeutic index relative to the unconjugated
alkylating agent.10,25
During the past decade, the biosynthetic machinery of natural
polyamines (Figure 1) has emerged as a major target for
anticancer therapy.1,2 A variety of inhibitors of polyamine
biosynthetic enzymes and, more recently, of polyamine ana-
logues capable of interfering with the functions of naturally
occurring polyamines have been designed.3,4 Some potent
antineoplastics are actually in clinical trials. Other strategies
have led to the development of drugs conjugated to a polyamine
moiety.4,5 The idea was to use polyamine properties that could
potentially benefit the activity or the specificity of drugs. Most
designed conjugates tried to take advantages of two features of
natural polyamines: (i) their affinity for nucleic acids6 and (ii)
the existence of specific cell polyamine transport systems.7,8
Other properties of polyamines such as their affinity for
ionotropic glutamate and nicotinic acetylcholine receptors, which
lead to the design of potent antagonists,9 will not be discussed
here.
Conjugation of polyamines to a variety of nucleic acid
targeting drugs, such as DNA intercalators, alkylating agents,
or DNA- or RNA-cleavers has been reported.10-21 Tethering
spermine to a DNA-binding agent increases considerably the
overall DNA binding affinity as expected from the synergistic
activity of two DNA ligands.11,20,22,23 The conjugation of the
aromatic nitrogen mustard chlorambucil to spermidine enhances
10 000-fold its ability at forming interstrand cross-links with
naked DNA. However, the activity of these conjugates greatly
depends on their architecture: for example, the attachment of a
Alternatively, the polyamine moiety may confer DNA-binding
affinity to compounds devoid of such affinity, as demonstrated
for boron- or fluorine-containing N-benzylspermidine derivatives
designed, respectively, for boron neutron capture therapy and
positron emission tomography.26 The high affinity of polyamines
for DNA has been also used to design DNA delivery agents
for nonviral gene therapy as recently reviewed.27
Targeting the polyamine transport system (PTS) is another
reason for designing drug-polyamine conjugates. All cells
possess active and energy-dependent PTS, which take up
exogenous natural polyamines but also structurally related
compounds.7,8 Because of their high demand for polyamines,
tumor cells have higher PTS activity than normal cells.28-30
Consequently, a drug-polyamine conjugate would accumulate
preferentially in the tumor cells if it can be taken up by the
PTS. Its accumulation could be boosted by depleting intracellular
polyamine pools [e.g., with R-difluoromethylornithine (DFMO),
an inhibitor of ornithine decarboxylase], which results in the
upregulation of the PTS activity.8 Thus, vectorization of
chemotherapeutic drugs by polyamines, made possible by the
broad structural tolerance of the PTS, is expected to enhance
cytotoxicity to tumor cells and to diminish secondary effects
on normal cells.4,7 In contrast, another approach aims at
inhibiting the PTS, and some polyamine conjugates have been
reported to be potent PTS inhibitors.31-35 Inhibition of polyamine
transport with specific antagonists may potentiate the therapeutic
action of DFMO under in vivo conditions where exogenous
polyamines tend to replenish cellular pools.3 This inhibitory
* Corresponding author: phone 33-(0)2-23-23-45-03; fax 33-(0)2-23-
23-44-78; e-mail delcros@univ-rennes1.fr.
† Groupe de Recherche en The´rapeutique Anticance´reuse, Faculte´ de
Me´decine, Universite´ Rennes 1.
‡ Groupe Cycle Cellulaire, CNRS UMR 6061 “Ge´ne´tique et De´veloppe-
ment”, IFR140 GFAS, Faculte´ de Me´decine, Universite´ Rennes 1.
§ Substances liche´niques et photoprotection, EA, Institut de Chimie de
Rennes, Faculte´ de Pharmacie, Universite´ Rennes 1.
| Laborato´rio de Sintese de Fa´rmacos, Faculdade de Farma´cia/UFRGS.
10.1021/jm050018q CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/03/2005

Polyamine Homologation of Heterocyclic Amidines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 233
activity has to be considered for the design of polyamine
conjugates conceived to be selectively uptaken by the PTS, since
such inhibitory activity may severely impair their uptake via
the polyamine transporter.20
To date, significant work has been accomplished in the
characterization of PTS in bacteria, yeast, and, very recently,
protozoan parasites.36-39 However, in mammals, despite recent
advances on the mechanism of polyamine transport,40,41 no
proteins have yet been characterized. The lack of molecular
information has hampered the rational design of PTS-selective
polyamine-drug conjugates, and misconceptions (e.g., confu-
sion between ability to inhibit the uptake of natural polyamines
and ability to serve as a substrate) have slowed progress.
Investigations on the selectivity of drug-polyamine conjugates
toward the PTS require straightforward approaches such as the
comparison of their accumulation in wild-type and polyamine-
transport-deficient cell lines.26,42-44 If methods to determine
levels are not available, comparison of the cytotoxicity of
conjugates on both type of cells may be considered as an
alternative.20,45-49 Because polyamine depletion leads to an
enhancement of the PTS activity, the comparison of the
accumulation of conjugates in normal and polyamine-depleted
cells is also a reliable approach.26,45,49-51 In contrast, inaccurate
conclusions may be drawn from the comparison of cytotoxicity
of conjugates on normal and polyamine-depleted cells. Indeed,
if polyamine depletion does enhance the cytotoxicity of PTS-
selective drugs (due to their more efficient uptake), it also
improves the cytotoxicity of certain polyamine-unrelated che-
motherapeutic agents. In particular, DFMO-induced polyamine
depletion has been shown to potentiate the cytotoxicity of DNA-
targeting agents, such as BCNU, an effect that has been assigned
to chromatin structure alterations resulting from polyamine
loss.6,52
Figure 1. Structures of naturally occurring putrescine (1), spermidine
(2), and spermine (3).
nitrogen substituted by alkyl-, hydroxylalkyl- or aminoalkyl-
bearing side chains. These conjugates, which exhibited signifi-
cant in vitro cytotoxicity on ovarian carcinoma cells and in vivo
antitumor activity when given in association with DFMO,54 were
found to possess weak but significant DNA-binding affinity,
which correlated with their in vitro cytotoxicity.55 The putrescine
conjugates (4b, 5b) that exhibited the greater affinity for DNA
were around 10-fold more cytotoxic than their alkyl or hy-
droxylalkyl homologues. As discussed above, two reasons may
explain the potentiation of these conjugates in the presence of
DFMO: (i) their putrescine moiety may provide the ability to
use the polyamine transport system and then to accumulate to
a greater extent in polyamine-depleted cells, and (ii) depletion
of polyamines may affect chromatin structure and increase the
accessibility of the conjugates to DNA.
To further extend our knowledge of the structure-activity
relationships (SAR) of polyamine conjugates with complex
systems, we synthesized two sets of the nonplanar tetracyclic
systems 4 and 5 conjugated with spermidine 2 (SPD) or
spermine 3 (SPM). The substitution of the putrescine side chain
by higher polyamines may improve the activity of the amidine
derivatives, since these polyamines have higher affinity for the
PTS as well as for DNA. Three other sets of polyamine
conjugates were obtained by simplification of the initial
polycyclic structures 4 and 5, leading to sets 6 and 7, and the
3,4-dihydroquinoline derivatives (8).
Systematic studies appear crucial to identify molecular
recognition elements and to delineate the structural tolerance
accommodated by the PTS. Two collections of polyamine
conjugates were synthesized and screened for affinity for the
PTS and uptake via the PTS.
Our aim was to determine whether these new polyamine
conjugates were selective substrates of the PTS and therefore
possessed properties required for tumor targeting. For this
purpose, their affinity for PTS was determined in L1210 cells
as well as their selective cytotoxicity and accumulation in cells
with active PTS. In addition, our investigations were extended
to other biological properties that may be altered by the presence
of the polyamine chain, such as DNA-binding and calmodulin
antagonism (both activities that may influence the drug cyto-
toxicity).
The first collection consisted of several N-substituted linear
or branched di-, tri-, or tetramine scaffolds with different tether
lengths between nitrogen centers. The N-substituents were N1-
alkylarenes, with the alkyl spacer ranging from methyl to propyl
and the arene nucleus from benzene to pyrene.47-49,53 These
conjugates helped define some key characteristics required for
the selective delivery of polyamine-drug conjugates into cells
with active polyamine transporters. The triamine homospermi-
dine appeared to be a highly selective vector motif to ferry an
arenyl group into cells via the PTS.48,53 Tetraamines confered
high PTS affinity for conjugates but this did not translate into
a more efficacious drug.47 The PTS can accommodate relatively
large N1-substituents such as a pyrene nucleus.53 The length of
the tether connecting the polyamine chain to the arenyl group
is critical.49 These results lead us to define a model that describes
a hydrophobic pocket of set dimensions (able to accommodate
the N1-substituent) adjacent to a polyamine-binding site.49
Results and Discussion
Synthesis. Five different heterocycles were coupled by an
amidine function to 3-amino-1-propanol or to the natural
polyamines (putrescine, spermidine, and spermine), leading to
five sets of compounds (Figure 2). Starting from two tetracyclic
sets constituted by a 3,4-dihydroquinoline ring fused with a cis
junction either to a 2,3,4,5-tetrahydro-1H-benzazepine (4) or
to a 1,2,3,4-tetrahydronaphthalene (5), three other sets were
obtained by a disjunctive approach: sets 6 and 7 and the 3,4-
dihydroquinoline set (8). Thus, we have prepared two tetracyclic
sets either with an endocyclic tertiary amine (4a,b,d) or with
an exocyclic secondary amine (5b-d) and their opened
analogues either with a tertiary amine (6a-d) or with a
secondary amine (7b-d) and a bicyclic set (8a-d).
The second collection of polyamine conjugates, and the
subject of this report, comprised five sets of heterocyclic
amidines of various size and complexities conjugated to
aminopropanol or to the naturally occurring polyamines pu-
trescine (1; PUT), spermidine (2; SPD), or spermine (3; SPM)
(Figure 1). The design of these conjugates was based on previous
reports describing amidine derivatives of nonplanar tetracyclic
systems (Figure 2): tetrahydroquino[4,3-b][1]benzazepine (4)
and tetrahydrobenzo[k]naphthyridine (5), with their exocyclic
The synthesis of compounds 4-8 involved the thionation of
lactams 12-16, leading to 17-21, which were conjugated by
an amidine function to various amines. The starting lactams 12/

234 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Delcros et al.
Figure 2. Structure of polyamine-heterocyclic amidine conjugates 4a-8d.
Scheme 1a
a Reagents: (i) CH3OH/CH2Cl2; (ii) 2 M aqueous HCl, CHCl3, 80 °C; (iii) 3 M aqueous HCl, refluxing; (iv) Lawesson’s reagent (0.7-4 equiv) in
refluxing toluene or dioxane; (v) (CH3)3SiI, anhydrous CH2Cl2, room temperature; (vi) (CH3)3SiI, anhydrous CHCl3, 70 °C.
13 and 14/15 (Scheme 1) were obtained by rearrangement, in
acidic medium, of the corresponding 4-aminoazetidin-2-ones 9
and 11, respectively.56,57 The tetracyclic lactams 12 and 13,
bearing three chirality centers (C-6a, C-8, and C-13a or C-12b),
were obtained as a 60/40 (trans/cis) mixture of diastereomers.57
CH3-6a and CH3-8 were in a trans relationship in the major
isomers and in a cis relationship in the minor isomers. Because
the chromatographic separation of the diastereomers remained
unsuccessful, a chemical method was used to prepare pure or
enriched isomers. This method involved the formation of the
intermediate aminoethers 10r and 10â,56 which could be
separated by recrystallization in MeOH54 (Scheme 1). The
aminoether 10r was used for the synthesis of trans compounds
5b-d and 4a (enriched at 88%), and the aminoether 10â was
used for the synthesis of cis compound 4d (Figure 2). Compound
4b was used as a 40/60 (trans/cis) mixture of diastereomers. In
fact, in a previous study, we had shown that the diastereomers
of each tetracyclic set (4b, 5b) coupled to the putrescine in pure
forms or in mixture had the same order of activity on 3LL
carcinoma cells.54 Lactam 16 was purchased from Aldrich.
Compounds 12-16 contained an amide function whose
thionation (Scheme 1) was easily achieved according to a

Polyamine Homologation of Heterocyclic Amidines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 235
Scheme 2a
of regioisomers. Thus we have prepared N4,N8-di-(tert-butoxy-
carbonyl)spermidine according to a known method60 with the
following modification: the reduction of nitrile leading to the
formation of the terminal amino group was performed by
catalytic hydrogenation (H2/Ni Raney, EtOH/NH3) instead of
LiAlH4. We used it in smaller excess (5 equiv) for the coupling
step to the thiolactams. These spermidine conjugates (5-8c)
were chromatographed and then deprotected in acidic medium
(1 M aqueous HCl).
All conjugates 4a-8d (Figure 2) were purified by column
chromatography and transformed to hydrochlorides by use of
2 M HCl gas in ethanol. They were obtained in acceptable
overall yields (19-88%). Purity was checked by combustion
analysis (except 4a and 8a) and HPLC by a modified version
of a method reported for the determination of the natural
polyamines in biological samples.61 Generally, NMR spectra
were carried out in CDCl3 on the free base form. Because of a
coalescence problem, NMR spectra of 4d, 5d, 6d, 8c, and 8d
were performed on the hydrochloride salts in D2O. In addition,
the 13C NMR spectrum of 4a was performed in D2O.
a Reagents: (i) amine or protected amine excess, HgCl2 in refluxing THF;
(ii) 2 M HCl gas, ethanol.
classical method: the use of Lawesson’s reagent (0.7-4 equiv)
in refluxing dioxane or toluene led to the thiolactams 17-21
in good yields (46-87%), as was previously reported for 17
and 18.54
DNA-Binding Properties. The loss of ethidium fluorescence
as a consequence of the displacement of the DNA-bound
fluorochrome (at a high [ethidium]:[DNA] molar ratio) as a
function of added drug allows a rough estimation of the affinity
of drug binding to DNA.62-64 The drug concentration producing
50% inhibition of fluorescence (CC50) is approximately inversely
proportional to its DNA binding constant. CC50 values deter-
mined for free or conjugated polyamines are listed in Table 1.
In a previous report, the nonplanar tetracyclic conjugates 4a,
4b, and 5b have been found to possess weak but significant
DNA-binding affinity by thermal denaturation.55 In perfect
agreement with this report, we found that the tetrahydrobenzo-
naphthyridine 5b shows a greater overall DNA-binding affinity
than its isomeric counterpart 4b and that the introduction of an
The formation of an amidine bond from a thiolactam and an
amine is an efficient method,55,58,59 in which the presence of
mercury chloride (HgCl2) favors the completion of the reaction
by precipitation of mercury sulfide (HgS) (Scheme 2). In this
work, two different cases arose depending on the structure of
the amine moiety. The symmetrical polyamine putrescine and
spermine (as well as the amino alcohol) could be used without
any protective groups: their primary amino group reacted
predominantly and a large excess (10-25 equiv) avoided any
significant formation of bis-conjugates. Their large water
solubility allowed their removal by simple washing of organic
layers during the final workup. In contrast, the unsymmetrical
spermidine required selective protection to prevent the obtaining
Table 1. Various Biological Properties of the Polyamine-Amidine Conjugatesa
calmodulin
DNA binding properties, antagonism,
BSAO oxidation,
PUT transport
SPD transport
SPM transport
compd
CC50b (drug/DNA P)
IC50c (µM) rel fluorescence intensityd inhibition,e Km/Ki (µM) inhibition,e Km/Ki (µM) inhibition,e Km/Ki (µM)
AP
1
2
0
>50
>500
>500
>500
3 ( 0.1
19.9
0.75 ( 0.07
0.26 ( 0.03
5.5( 0.6
0.28 ( 0.01
85 ( 4
2.45 ( 0.51
1.34 ( 0.31
3
125 ( 5
1.06 ( 0.02
0.24 ( 0.02
4a
4b
4d
>50
23 ( 1
3.2 ( 0.3
>500
0
0
106 ( 7
116 ( 10
29.2 ( 5.4
19 ( 4
42 ( 2
0.058 ( 0.006
0.30 ( 0.05
1.01 ( 0.21
5b
5c
5d
7.8 ( 0.1
2.5 ( 0.2
1.6 ( 0.3
70 ( 11
22 ( 5
10 ( 6
0
8.20 ( 1.03
2.54 ( 0.20
0.56 ( 0.06
13 ( 0.4
67 ( 1
6a
6b
6c
6d
>500
0
15 ( 1
5.1 ( 0.1
2.2 ( 0.1
1.8 ( 0.2
51 ( 9
30 ( 12
8 ( 4
5 ( 0.1
17 ( 0.8
53 ( 1
20.1 ( 2.3
6.80 ( 0.52
0.052 ( 0.05
0.26 ( 0.04
0.20 ( 0.03
0.28 ( 0.04
7b
7c
7d
12.4 ( 0.4
1.8 ( 0.1
0.64 ( 0.16
373 ( 86
64 ( 5
23 ( 4
0
>150
5 ( 0.1
52 ( 1
3.41 ( 0.22
0.38 ( 0.02
8a
8b
8c
8d
5.5 ( 0.08
>500
0
0
>500
>500
>500
400 ( 52
1.58 ( 0.14
0.38 ( 0.03
1.19 ( 0.06
0.11 ( 0.01
46 (2
106 ( 1
0.75 ( 0.05
0.20 ( 0.01
a All data are expressed as mean ((SD) from three separate determinations. b Conjugate concentration to give 50% decrease in fluorescence of calf
thymus DNA-bound ethidium. c Concentration of conjugate required to inhibit by 50% c-AMP-dependent calmodulin-activated phosphodiesterase. d The
hydrogen peroxide generated by the oxidation of the test compound (10 µM) in the presence of bovine serum oxidase was determined by the peroxidase-
catalyzed transformation of homovanillic acid into a fluorescent product. e Polyamine uptake inhibition was determined in L1210 cells. Km values for polyamine
uptake (shown in boldface type) were determined by Lineweaver-Burk analysis of transport velocity at increasing radiolabeled substrate concentrations. Ki
values were calculated from the half-maximal inhibitory concentration (IC50) estimated by iterative curve fitting for sigmoidal equations describing polyamine
uptake velocity in the presence of growing concentrations of antagonist.

236 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Delcros et al.
aminoalkyl side chain (putrescine) favors interaction over a
hydroxylalkyl side chain. It has to be noticed that 6a has a higher
affinity for DNA than its closed counterpart 4a and that the
“naked” dihydroquinoline 8a had a similar activity as 6a.
three positive charges (IC50 between 20 and 60 µM). In turn,
the latter were more potent than the putrescine conjugates with
only two positive charges (IC50 between 50 and 370 µM).
Similarly, the spermine sulfonamides, with one positive charge
of the spermine moiety lost by amide bond formation with a
sulfonic acid,44 had similar potency as spermidine conjugates
5-7c. Because of their calmodulin antagonism, some conjugates
may limit their own active uptake via the polyamine transport
system.
The CC50 values determined for free polyamines are in
agreement with earlier studies65-68 that have clearly demon-
strated a relationship between the number of protonated nitro-
gens of free polyamines and their affinity for nucleic acids. The
affinity for DNA varied as follows: spermine (tetramine) >
spermidine (triamine) > putrescine (diamine). A similar rela-
tionship was also observed for the conjugated polyamines (4-
8), with the highest affinity observed for spermine conjugates.
Increasing the number of amino groups on the side chains in
all series enhanced the affinity for double-stranded DNA. As
expected from the additive or synergistic activity of two ligands
with DNA affinity,11,20 putrescine and spermidine conjugates
were more efficient to release bound ethidium than their free
forms. Surprisingly, free spermine remains more efficient than
its conjugated forms. It has been suggested that the tetracyclic
conjugates 4a, 4b, and 5b bind only weakly to DNA via the
minor groove rather than through an intercalative process.55 Due
to the much higher affinity of spermine for DNA, the binding
of spermine conjugates may be completely driven by the
polyamine side chain and may be constrained to natural
“polyamine” binding sites. In this hypothesis, a critical factor
may be the bulkiness of the heterocyclic moiety, which may
hinder spermine binding to DNA. In agreement with such
hypothesis, the DNA-binding affinity of the spermine conjugates
varies inversely with substituent size: for example, the affinity
for DNA of the tricyclic 6d was higher than that of the
tetracyclic conjugate 4d but lower than that of the bicyclic
conjugates 8d.
Oxidation by Serum Amine Oxidase. Bovine serum amine
oxidase (BSAO, EC 1.4.3.6) catalyzes the oxidation of sper-
midine and spermine, giving rise to the corresponding aldehydes,
ammonia, and hydrogen peroxide.77,78 While aminopropanol and
putrescine derivatives showed no oxidation by BSAO, all
spermidine and spermine conjugates were oxidized, as evidenced
by the formation of a fluorescent adduct from homovanillic acid
in the presence of peroxidase (Table 1). However, the conjugates
were poorer substrates than the free corresponding polyamines.
Because the oxidation products are cytotoxic, cell culture
experiments were performed in the presence of aminoguanidine,
an inhibitor of BSAO.
Cell Studies. Three cell lines were chosen for bioassay.
L1210 (mouse leukemia) cells were selected to enable com-
parisons with the published IC50 and Ki values known for a
variety of polyamine substrates.53,79-81 CHO cells were chosen
along with a mutant cell line (CHO-MG) in order to comment
on selective transport via the PTS.20,47
Cytotoxicity on L1210. As shown in Table 2, all conjugates
displayed moderate or no cytotoxicity on L1210 cells. The
introduction of a (poly)aminoalkyl side chain influenced the
cytotoxicity of the compounds. Putrescine derivatives 4b, 5b,
and 6b were potent agents with IC50 below 32 µM and were
more active than their aminopropanol counterparts, while 7b
and 8b were completely devoid of any activity. The introduction
of a spermidine side chain was not beneficial since none of the
spermidine conjugates had any effect on L1210 cell growth. In
contrast, all spermine derivatives were cell growth inhibitors.
The position of the methylamino substituent had a critical impact
on the cytotoxicity of the compounds. Compounds with the
methylamino substituent remote from the amidine residue (5d,
7d; IC50 ) 28 µM) were more potent than compounds 4d and
6d. It should be noted that 8d, a compound devoid of any
substituent, had an IC50 around 70 µM.
Calmodulin Antagonism. Calmodulin is a ubiquitous Ca2+
-
binding protein involved in multiple vital cell functions,69 and
calmodulin antagonists are cytotoxic agents.70 Spermine 3 is a
weak calmodulin antagonist,71 but polyamine conjugates44 as
well as other analogues72 have been reported to be very potent
calmodulin antagonists, a property that could contribute to their
cytotoxicity.4,73 In addition, calmodulin activation has been
shown to be involved in the regulation of polyamine uptake,
and calmodulin antagonists are known to inhibit polyamine
uptake.74-76 We then investigated the ability of our polyamine
conjugates to inhibit calmodulin-activated cyclic nucleotide
phosphodiesterase (Table 1).
We did not observe any correlation between the cytotoxicity
of the drugs and either their DNA binding or their calmodulin
antagonism properties. This could be related to their ability to
be taken up by cells.
Unconjugated polyamines 1, 2, and 3 had very little effect
on calmodulin-activated cyclic nucleotide phosphodiesterase
with IC50 greater than 0.5 mM. In contrast, all polyamine
conjugates of sets 4-7, but not their aminopropanol analogues,
inhibited calmodulin-activated cyclic nucleotide phosphodi-
esterase. Because none of the conjugates had any effect on basal
phosphodiesterase activity (data not shown), their inhibitory
effect on the calmodulin-activated enzyme is likely due to their
interaction with calmodulin. Spermine conjugates were more
potent antagonists with IC50 below or equal to 23 µM. Spermine
is known to bind calmodulin but its binding affinity is in the
millimolar range.71 It has been postulated that these latent
properties of spermine may be “amplified” by conjugation with
a substituent of significant lipophilicity.44 In support of this
assertion is the observation that polyamines conjugated to the
less hydrophobic dihydroquinoline system (set 8) were devoid
of calmodulin antagonism. It is also obvious that the number
of positive charges contributes to the calmodulin antagonist
properties. The spermine conjugates 4-7d with four positive
charges were more potent than the spermidine conjugates with
PTS Selectivity. To comment on selective transport via the
PTS, we determined for each conjugate: (i) the Ki value (Table
1), which reflects its affinity for the PTS, in a competitive assay
with radiolabeled polyamines in L1210 cells; (ii) its growth
inhibitory effect (Table 2) and accumulation (Table 3) in CHO
and polyamine transport-deficient CHO-MG cells; and (iii) the
effect of DFMO on its L1210 cell growth inhibitory effect
(Table 2) and cellular accumulation (Table 4).20,47,48,53
(i) All polyamine conjugates were determined to be competi-
tive inhibitors of the PTS against at least one of the three natural
polyamines 1-3. The affinities of free and substituted polyamines
for the PTS in L1210 cells are listed in Table 1. As expected
from earlier work,47,48,53,82 the affinity for the L1210 polyamine
transporter increased with the number of nitrogen centers. The
affinity varies as follows: tetramines > triamines > diamines
> monoamines. The spermine derivatives demonstrated very

Polyamine Homologation of Heterocyclic Amidines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 237
Table 2. In Vitro Cell Growth Inhibitiona
IC50b (µM)
CHO
L1210
CHO-MG
compd
control
+DFMOc
control
+DFMO
rev
rev
rev
control
+DFMO
AP
1
2
>100
>100
>100
>100
revd (>0.5 µM)
rev (>0.1 µM)
rev (>0.1 µM)
>100
>100
>100
>100
>100
>100
>100
>100
>100
3
4a
4b
4d
100
32
78
54 ( 4e
26 ( 1
21 ( 1
45 ( 3e
9.1 ( 1e
26 ( 3
24 ( 1
24 ( 2
27 (3
1.0 ( 0.1e
5b
5c
5d
28
>100
22
56 ( 4
31 ( 2
>100
23 ( 2
57 ( 4e
33 ( 2
>100
29 ( 1
30 ( 1
>100
23 ( 2
rev (>10 µM)
rev (>2 µM)
2.4 ( 0.2e
11 ( 2e
6a
6b
6c
6d
>100
15
>100
68
33 ( 2e
13 ( 1
>100
>100
rev (>1 µM)
14 ( 1
>100
13 ( 2
>100
rev (>5 µM)
2.3 ( 0.2e
7b
7c
7d
>100
>100
28
rev (>10 µM)
rev (>5 µM)
3.1 ( 0.2e
>100
>100
23 ( 3
>100
>100
30 ( 2
11 ( 1e
23 ( 1
8a
8b
8c
8d
>100
>100
>100
>100
rev (>5 µM)
>100
rev (>0.1 µM)
>100
>100
>100
>100
>100
>100
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 by a MTT assay after 48 h of treatment. b Conjugate concentration required to inhibit cell growth by 50% after 48 h; mean value ((%) from
three determinations. c DFMO (5 mM) was added at the time of the addition of the conjugates. d Reversion; compound that reverses the cytostatic effect of
DFMO at concentrations above that indicated in parentheses. e p < 0.05: significantly different from control values without DFMO.
Table 3. Cellular Uptake of Conjugates in CHO and CHO-MG Cells
cellular uptakea (nmol/mg of protein)
compd
concn (µM)
CHO
CHO-MG
CHO/CHO-MG ratio
4b
4d
25
50
3.6 ( 0.8
0.66 ( 0.10
3.3 ( 0.6
0.66 ( 0.11
1.1
1.0
5b
5c
5d
25
50
25
8.1 ( 0.5
1.3 ( 0.2
2.2 ( 0.1
7.2 ( 1.3
1.2 ( 0.5
2.1 ( 0.2
1.1
1.0
1.0
6b
6c
6d
10
25
50
13 ( 2
2.9 ( 0.1
3.3 ( 0.08
12 ( 2
3.1 ( 0.7
3.2 ( 0.3
1.1
1.0
1.0
7b
7c
7d
50
50
25
5.3 ( 0.5
1.2 ( 0.3
0.72 ( 0.2
5.9 ( 0.2
1.7 ( 0.1
0.81 ( 0.3
0.9
0.7
0.9
8b
8b
8c
8c
8d
8d
5
10
10
25
5
1.2 ( 0.1
2.1 ( 0.5
11.0 ( 2.0
15.4 ( 1.5
0.029 ( 0.004
0.029 ( 0.002
0.26 ( 0.04b
0.37 ( 0.05b
1.6 ( 0.2b
4.6
5.7
6.9
7.0
1.0
1.3
2.2 ( 0.3b
0.029 ( 0.003
0.022 ( 0.004
10
a Conjugates were added 24 h after seeding; intracellular levels were determined by HPLC on perchloric extracts of cells treated with the conjugates
during 24 h; mean values ((SD) from three determinations are given. b p < 0.05: significantly different from values determined in CHO cells.
high affinity with Ki values in the nanomolar range. The affinity
for the PTS was essentially driven by the polyamine moiety.
spermidine conjugates 8b and 8c were up to 7 times higher in
CHO than CHO-MG cells, indicating that the PTS is involved
in their uptake. No differential accumulation was observed for
their spermine homologue 8d, which suggests that spermine is
not a PTS-selective vector.
(ii) Comparison of cytotoxicity in CHO and CHO-MG cells
provides an important screen to detect selective conjugate
delivery via the PTS.20,47,48,53 A clear limit to this assay is the
cytotoxicity of the drugs and in the present study; we were
unable to determine IC50 values for seven out of the 13
compounds assayed. Yet none of the six polyamine conjugates
(with a measurable growth inhibitory effect) showed any
selectivity on CHO. To overcome the cytotoxicity issue, we
determined the intracellular concentrations of the conjugates in
both cell lines (Table 3). Conjugates of sets 4-7 did not show
any differential accumulation in CHO vs CHO-MG cells. In
contrast, the intracellular concentrations of the putrescine and
Because putrescine and spermidine conjugated to bulkier
substituents (e.g., 4-7b,c) were not taken up by the PTS, we
may conclude that there are limits to the size of substituents
which can be accommodated by the PTS. A large substituent
would not fit in the hydrophobic pocket of set dimensions
adjacent to the PTS polyamine binding site modeled from our
recent study on N1-arylalkylpolyamines containing various
aromatic ring systems.49 Spermidine imparts selectivity of small
substituents, such as a benzyl moiety26 or N-methylanthranylic

238 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Delcros et al.
Table 4. Polyamine and Analogue Levels in L1210 Cells Cultured for 48 h in the Presence of Amidine Derivatives, in the Absence or Presence of
DFMO
polyamine levelsa (nmol/mg of protein)
PUT
SPD
SPM
conjugate
compd
concn (µM)
-DFMO
+DFMO
-DFMO
+DFMO
-DFMO
+DFMO
-DFMO
+ DFMO
0
20
10
1.6
0.85
1.5
0.05
0.70b
0.04
22
11c
18
0.3
8.8
6.5
9.0b
5.8
4b
4d
0.87b
0.49
6.3c
9.7c
13
0.59
8.2d
1.5d
5b
5c
5d
20
25
10
1.6
1.0
1.4
1.0b
0.02
0.04
14c
24
2.1b
1.5b
0.51
8.9
11c
8.2
10b
23
1.2
0.95
8.2d
1.3
8.9b
7.3b
16c
2.1d
6c
6d
25
10
0.65c
0.41c
0.12
0.02
21
1.4b
10c
10b
0.64
1.8
0.66
2.6d
16c
0.60b
9.5c
7.6b
7b
7c
7d
50
50
10
1.9
1.1
1.1
1.0b
0.08
0.07
20
15c
17c
1.8b
8.6
6.6
0.60
0.77
0.54
0.33d
0.62
2.4b
9.2c
9.4c
8.0b
7.8b
0.66b
0.74d
8b
8c
8d
10
10
10
1.8
bdl
bdle
bdl
bdl
21
0.94b
3.2b
12.5c
7.1
16.5b
12.1b
13.9b
0.26
4.3
1.5
0.34d
8.2d
14.5c
8.1c
0.33c
4.2b
13.9c
2.4d
a Conjugates and DFMO (5 mM) were added at the time of seeding; intracellular levels of polyamines and conjugates were determined by HPLC on
perchloric extracts of cells cultured for 48 h. b Significantly different between treated and control cells (p < 0.05). c Significantly different between treated
and control cells in the presence of DFMO (p < 0.05). d Conjugate levels significantly different between cells treated in the absence or in the presence of
DFMO (p < 0.05). e bdl, below detection levels.
acid,83,84 as well as more bulky tricyclic cargos such as
BODIPY, a fluorescent probe,41,83 or anthracene.48 It was not
surprising that spermidine was also a selective motif to ferry
the bicyclic dihydroquinoline system (8c). For putrescine, the
bulkiness of the substituent is even more limiting. Putrescine
is a selective vector to ferry benzyl26 and dihydroquinoline but
not anthracene systems.53 These observations demonstrate that
the limits to the size of substituents depend also on the nature
of the polyamine chain.
PTS. Multiple systems for the uptake of polyamines and related
compounds with different characteristics have already been
described in a given cell line.87,88 The synergistic effect observed
with the amidine spermine conjugates, which is similar to that
reported for amidoacridine-spermine conjugates,20 could be
explained by the presence of a high-affinity PTS expressed only
in DFMO-treated cells. Such a PTS would be less stringent than
the one expressed in normal L1210 or CHO cells since it will
allow the transport of bulky conjugates.
(iii) Inhibition of ornithine decarboxylase by R-difluoro-
methylornithine (DFMO) leads to a significant increase in
polyamine uptake as a reaction to the depletion of intracellular
polyamine pools.85,86 Therefore, cytotoxic polyamine conjugates
that selectively target the PTS should be more potent on DFMO-
treated cells.
In contrast, all putrescine and spermidine conjugates antago-
nized the effect of DFMO in L1210 cells as shown in Table 2.
They reversed the cytostatic effect of DFMO at low concentra-
tions (e.g., 5b in Figure 3), and the potency of the cytotoxic
conjugates such as 4b, 5b, and 6b was significantly diminished.
In L1210 cells treated with the putrescine or spermidine
conjugates in the presence of DFMO, intracellular levels of free
putrescine and/or spermidine were significantly higher than in
DFMO-treated cells not exposed to the drugs (Table 4). One
possible explanation for these observations is the release, from
the conjugates, of free putrescine or spermidine, which will in
turn antagonize the DFMO-induced cytostatic effect. Release
of free polyamine from various polyamine conjugates has
already been reported, especially in DFMO-treated cells.26,49,89
However, the reversion of the DFMO-induced cytostatic effect
was observed in CHO but not in CHO-MG cells (Table 2, Figure
3), indicating that the reversion was entirely dependent on an
active PTS. This strongly suggests that the release of free
polyamines from the conjugates occurred outside the cells in
the culture medium. However, after incubation for 48 h at 37
°C of putrescine conjugates in culture medium, only traces of
free putrescine were detected (data not shown). It is then possible
that cultured cells released into the extracellular space an enzyme
capable of degrading the conjugates. This remains to be
investigated.
In L1210 and CHO cells, but not CHO-MG cells, DFMO
increased the potency (lower IC50 values) of all spermine
conjugates, except the noncytotoxic compound 8d (Table 2).
For spermine conjugates 4-7d, the growth inhibitory curves
showed a strong synergistic effect at low concentrations, as
shown for conjugate 4d in Figure 3. As discussed above, this
synergistic effect could be due to the intrisinc activity of DFMO.
However, the synergistic effect was not observed in DFMO-
treated CHO-MG cells. Because CHO-MG cells do not show
any alteration in polyamine metabolism and regulatory pathways
besides the lack of a functional PTS,86 the absence of synergistic
effect in DFMO-treated CHO-MG cells suggests that the
synergy was entirely dependent on an active PTS. The deter-
mination of intracellular levels of conjugates in L1210 reveals
that polyamine-depleted cells accumulated more efficiently (up
to 2 times more) the spermine conjugates (included 8d) than
normal cells (Table 4). In L1210, the potency of the spermine
conjugates was similar when the cells were treated in the
presence of 200 µM spermidine (data not shown). If the
conjugates were using the PTS, spermidine would have been a
potent competitor and would have decreased the potency of the
drugs.49 Altogether, these results indicate that in normal cells
(referring to cells not treated with DFMO) the spermine
conjugates are taken up independently of any PTS. In contrast,
in DFMO-treated cells, the synergistic effect was likely the
consequence of a supplementary uptake of the conjugates via a
The polyamines released from the conjugates were eventually
converted into higher polyamines inside the cells, as indicated
by the elevated spermine levels in DFMO-treated cells exposed
to the putrescine and spermidine conjugates (Table 4). Spermine
and spermidine concentrations were also higher in DFMO-
treated cells when incubated with spermine conjugates (Table

Polyamine Homologation of Heterocyclic Amidines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 239
Figure 3. Effect of the conjugates 4d (left panels) and 5b (right panels) on L1210 cell growth (upper panels) or CHO and CHO-MG cell growth
(lower panels) in the presence or absence of DFMO. Cells were cultured for 48 h with the conjugates in the presence or absence of 5 mM DFMO.
Cell growth rates were determined by the MTT assay. The relative cell growth rates were calculated from the value of cell growth of the corresponding
control cells cultured in the absence (OD540nm ) 1.14, 1.03, and 1.04 for CHO, CHO-MG, and L1210 cells, respectively) or in the presence of
DFMO (OD540nm ) 0.66, 0.65, and 0.61 for CHO, CHO-MG, and L1210 cells, respectively).
Figure 4. Spermidine uptake activity in CHO cells preincubated with various concentrations of spermine (3) or of the spermine conjugate 4d in
the presence or absence of 200 µM cycloheximide (CHX).
2). This could be explained by the release of free spermine from
the conjugates and its retroconversion into spermidine.
It is well established that, in response to an elevation of
intracellular polyamine pools (e.g., due to the transport of
extracellular polyamines), a regulatory pathway aimed at
preventing an excessive accumulation of polyamines, that may
be detrimental to the cells, is activated. This pathway involves
the de novo synthesis of antizyme, a protein responsible for
the inhibition and degradation of ODC that also negatively
regulates the PTS activity.8,91,92 As expected, the preincubation
of CHO cells with spermine 3 induced a dose-dependent
decrease in the spermidine uptake activity (Figure 4). This
decrease was prevented when spermine was applied in the
presence of 200 µM cycloheximide (CHX), an inhibitor of
protein synthesis. This is in agreement with an antizyme-
dependent downregulation of the PTS activity. A similar dose-
dependent decrease in PTS activity was observed in CHO cells
preincubated with 4d. However, the effect of 4d was insensitive
It should be mentioned that the treatment of L1210 cells with
DFMO enhanced the accumulation of the putrescine and
spermidine conjugates 8b and 8c, in agreement with their
selectivity for the PTS observed in the CHO/CHO-MG system.
Inhibition of the PTS Activity. Despite its high affinity for
the PTS (Table 1), the absence of selectivity of the spermine
conjugate 8d compared to its putrescine and spermidine
homologues (8b and 8c, respectively; Table 3) observed in CHO
vs CHO-MG cells indicates that the spermine moiety is not a
suitable motif for an efficient delivery via the PTS in the absence
of DFMO, in agreement with our previous reports.20,47 Because
some spermine conjugates are potent and transitory irreversible
inhibitors of the PTS,20,33,34,90 we envisaged that the spermine
conjugates may inhibit their own selective delivery via the PTS.

240 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Delcros et al.
tectural and physiochemical constraints allowed by the polyamine
transporter of mammalian cells. Our results clearly show that
spermidine as well as putrescine is a PTS-selective motifs, but
the size of substituents is a limiting factor for the conjugate
selectivity. In contrast, spermine, independent of the size of the
substituent, was effective in targeting the PTS for cellular entry
but only in cells treated with DFMO. Altogether, these findings
help further define the key characteristics of polyamine-drug
conjugates that can be accommodated by the polyamine
transporter.
Experimental Section
Chemistry. Reagent-grade solvents were purchased from chemi-
cal companies and used directly without further purification unless
otherwise specified. Tetrahydrofuran (THF) was dried under
nitrogen by distillation over sodium, and benzophenone and diethyl
ether were dried by distillation over LiAlH4. Dry ethanol was stored
over 4 Å molecular sieves.
Figure 5. Spermidine uptake activity in CHO cells preincubated with
spermine (3; 0.2 µM) or with the spermine conjugate 4d (0.25 µM) as
a function of the preincubation time.
Merck silica gel 60 (70-230 mesh) was used as the solid phase
for column chromatography. Thin-layer chromatographies were
performed on Merck silica gel 60 F254 (layer thickness 0.22 mm).
Solvent systems (expressed in volume percents) and Rf are indicated
in the text. The compounds were visualized with UV light,
ninhydrin, or iodine. FTIR spectra were recorded on a Perkin-Elmer
16PC instrument (KBr pellets, ν in reciprocal centimeters). NMR
spectra were recorded on a Bruker DMX spectrometer at 500 MHz
(1H) or 125 MHz (13C) except for 4a, 5c, and 6c for which spectra
were recorded on a Bruker AM300 FT spectrometer at 300 MHz
(1H) or 75 MHz (13C). Tetramethylsilane (TMS) was used as the
internal standard for NMR spectra performed in CDCl3. 3-(Trim-
ethylsilyl)-1-propanesulfonic acid (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 from chemical shifts, selective decoupling experiments,
and coupling constants (1J and long-range coupling). Values with
an asterisk can be inverted.
Electronic impact high-resolution mass spectra (HRMS EI) were
recorded on a Varian MAT 311 double-focusing instrument at the
CRMPO (Centre Re´gional de Mesures Physiques de l’Ouest) with
a source temperature of 140 °C, an ion accelerating potential of 3
kV, and ionizing electrons of 70 eV and 300 µA. High-resolution
mass spectra determined by liquid secondary ion mass spectrometry
(HRMS LSIMS) were performed on a ZabSpec Tof Micromass at
the CRPMO with a source temperature of 40 °C, an ion (Cs+)
accelerating potential of 8 kV, and mNBA (meta-nitrobenzylic
alcohol) as matrix.
HPLC analyses were conducted according to versions of a
previously described method.61 This method consists of the separa-
tion of amines as ion pairs with n-octanesulfonate. Reversed-phase
high-performance liquid chromatography (HPLC-1) was performed
on a C18 column (Nucleosil 5, C18 AB, 100 mm-5 µm) from
Macherey-Nagel (Du¨ren, Germany) on a SpectroPhysics Thermo-
quest HPLC instrument (for compounds 4a, 6a, and 8a) or on a
C18 column (Nucleodur, 150 mm-5 µM) from Macherey-Nagel
(Du¨ren, Germany) on a LDC/Milton Roy HPLC instrument (for
the other compounds). The solvent system used was the follow-
ing: buffer A, 0.1 N potassium acetate, pH 4.75, 5% acetonitrile,
and 10 mM n-octanesulfonate; buffer B, 0.1 N potassium acetate,
pH 4.75, 50% acetonitrile, and 10 mM n-octanesulfonate; gradient,
0 min (0% B); 1 min (0% B); 15 min (30% B); 27 min (50% B);
31 min (100% B); 38 min (100% B); 39 min (0% B); 45 min (0%
B); flow rate, 1 mL/min. A second reversed-phase HPLC (HPLC-
2) was performed on a C18 column (Nucleodur, 150 mm-5 µM)
from Macherey-Nagel (Du¨ren, Germany) on a LDC/Milton Roy
HPLC instrument with MeOH 20/buffer A 80 as solvent system
with a 1.5 mL/min flow rate. Absorbance at 254 nm was recorded
on a UV 2000 detector (Thermo Separation Products), and
fluorescence intensity (excitation at 345 nm; emission at 455 nm)
was recorded on a FL2000 fluorescence detector (Thermo Separa-
to the presence of CHX, as already reported for various spermine
conjugates such as acridine-spermine and lysine-spermine.20,33
These observations eliminate antizyme as an effector in the
downregulation of the PTS activity induced by these spermine
conjugates. All these spermine conjugates displayed a very high
affinity for the PTS as demonstrated by a very low Ki value
(<0.1 µM) for spermidine uptake (Table 1).
As discussed earlier, calmodulin, which is involved in the
regulation of polyamine uptake, may be envisaged as a potential
target of the spermine conjugates. Amidine-spermine conju-
gates were very potent antagonists with IC50 below or equal to
20 µM. Because of their calmodulin antagonism, some conju-
gates may limit their own active uptake via the polyamine
transport system. It has to be noted that 8b and 8c, the only
two compounds that displayed a preferential accumulation in
CHO versus CHO-MG cells, were lacking calmodulin antago-
nism. However, their spermine homologue 8d was also devoid
of this property but still did not show any selective uptake via
the PTS in CHO cells. Calmodulin antagonism may not be the
only mechanism limiting the selective uptake of polyamine
conjugates.
In CHO cells, the repression of the PTS activity induced by
spermine was time-dependent (Figure 5), as it is known to
require antizyme synthesis.8,91,92 In contrast, the repression of
the PTS activity induced by spermine conjugates such as 4d
was quite immediate after addition of the drug to the cells. As
shown in Figure 5, the drop of the PTS activity was already
maximal 2 min after the addition of 4d on CHO cells. The
rapidity of the repression induced by 4d suggests that the PTS
inactivation does not involve cell regulatory pathways but rather
implies a very rapid and high-affinity binding of the drug to
the transporter itself or to some effector. The proteoglycan
Glypican-1, recently identified as a possible vehicle for polyamine
uptake in mammalian cells, may be a possible target.40 Weeks
et al.33 have hypothesized that a slow rate of dissociation of
the conjugate from the transporter would prevent the recovery
of full transport activity after removal of the conjugates. In
agreement with such a supposition, we observed that an
anthracene-spermine derivative, a high-affinity PTS ligand,
binds rapidly to the cell surface and remains bound even after
extensive washing.47
Conclusion
Five sets of heterocyclic derivatives of various sizes and
complexities coupled by an amidine function to naturally
occurring polyamines were synthesized to challenge the archi-

Polyamine Homologation of Heterocyclic Amidines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 241
tion Products) after postcolumn derivatization with o-phthalalde-
hyde/N-acetylcysteine as previously described.61
Elemental analyses were performed by the Laboratoire de
Microanalyses (Faculte´ de Pharmacie, Universite´ Paris XI, Chaˆ-
tenay-Malabry).
Purity and characterization of compounds were established by a
combination of combustion analysis, HPLC, HRMS, and NMR
analytical techniques.
(CH2Cl2/EtOH/NH4OH 70/20/0.5, e), 0.17 (EtOH, f). HPLC-1 tR
32.35 min, 98.2% pure. HPLC-2 tR 27.31 min, 98.5% pure.
N-(3,3-Dimethyl-4-methylphenylamino-3,4-dihydroquinolin-
2-yl)-3-aminopropan-1-ol (6a). General procedure A from 19 (0.25
g, 0.85 mmol, 1 equiv) and 3-aminopropan-1-ol. Column chroma-
tography with CH2Cl2/acetone/NH4OH 80/20/0.5 (g). Oil; 39%; Rf
0.51 (g). HPLC-1 tR 31.91 min, 99.5% pure. Anal. (C21H27N3O,
HCl, H2O) C, H, N.
N-(3,4-Dihydroquinolin-2-yl)-3-aminopropan-1-ol (8a). Gen-
eral procedure A from 21 (0.25 g, 1.41 mmol, 1 equiv) and 3-amino-
1-propanol. Column chromatography with acetone/NH4OH 100/
0.5 (h). Oil, 52%; Rf 0.35 (h), 0.8 (acetone/NH4OH 100/0.1, i).
HPLC-1 tR 21.93 min, 98.5% pure. HPLC-2 tR 11.87 min, 98.5%
pure.
N-[3,3-Dimethyl-4-(methylphenylamino)-3,4-dihydroquinolin-
2-yl]putrescine (6b). General procedure A from 19 (0.200 g, 0.675
mmol, 1 equiv) and putrescine. Column chromatography with CH3-
OH/NH4OH 90/10 (j). Oil, 72%; Rf 0.43 (j). HPLC-1 tR 30.19 min,
99.5% pure. Anal. (C22H30N4, 2HCl, H2O) H, N; C calcd 59.86,
found 60.79.
The compounds were numbered for the heterocyclic moiety by
IUPAC rules and for the polyamine moiety by letters a-n (Figure
2).
Synthesis. Previously reported procedures were used for the
synthesis of compounds 12, 13, 17, and 18;54 14 and 15;57 4b;54,55
and 5b.55
General Procedure for Thionation. A mixture of lactam (1
equiv) and Lawesson’s reagent (0.7 or 1 equiv) was refluxed for 6
h in 60 mL of dry dioxane or dry toluene. After evaporation of
solvent under reduced pressure, the residue was purified by column
chromatography.
General Procedure A for Coupling Thiolactams to 3-Ami-
nopropan-1-ol, Putrescine, or Spermine. To a refluxing solution
of thiolactam (1 equiv) and 3-amino-1-propanol (10 equiv) or
putrescine (25 equiv) or spermine (15 equiv) in 2 mL of dry THF
was added HgCl2 (1 equiv). The mixture was refluxed for 1 h,
during which a black precipitate of HgS was observed. The THF
was removed under reduced pressure and the residue was suspended
in CH2Cl2. The mercury sulfide (HgS) was removed by filtration,
and then the organic layer was washed with 0.2 M aqueous Na2S2O3
to remove the unreacted HgCl2. After drying over K2CO3, the
organic layer was evaporated under reduced pressure. The residue
was purified by column chromatography.
General Procedure B for Coupling Thiolactams to the
Protected Spermidine. To a refluxing solution of N4,N8-di-(tert-
butoxycarbonyl)spermidine60 (5 equiv) and HgCl2 (1 equiv) in 2
mL of dry THF was added dropwise a solution of thiolactam (1
equiv) in 3 mL of dry THF over 45 min. A black precipitate of
HgS was observed. Refluxing was continued for 15 min under
stirring. The THF was evaporated under reduced pressure. The Boc
protective groups were removed by stirring the residue at 0 °C in
80 mL of 0.5 M aqueous HCl for 3 h. This aqueous solution was
then filtered to remove HgS, alkalinized with 32% aqueous NaOH,
and extracted with CH2Cl2. The organic layer was washed with a
0.2 M aqueous Na2S2O3, dried over K2CO3, and evaporated under
reduced pressure. The resulting crude residue was purified by
column chromatography with isopropylamine, CH3OH, and CHCl3
in a 1/4/4 ratio.
General Procedure for the Preparation of Hydrochlorides.
The amino conjugate was stirred in a 2 M solution of HCl gas in
ethanol (1.2 equiv/amino group). After evaporation of the ethanol,
the residue was triturated in anhydrous ether to give a white
hygroscopic solid.
3,3-Dimethyl-4-(methylphenylamino)-3,4-dihydroquinoline-
2(1H)-thione (19). General procedure for thionation from 14 (1.83
g, 6.52 mmol) and Lawesson’s reagent (0.7 equiv) in dry dioxane.
Column chromatography with CH2Cl2 (a). Yellow-orange solid,
58%; Rf 0.50 (a).
3,3-Dimethyl-4-(4-methylaminophenyl)-3,4-dihydroquinoline-
2(1H)-thione (20). General procedure fof thionation from 15 (1.24
g, 4.42 mmol) and Lawesson’s reagent (1 equiv) in dry dioxane.
Column chromatography with CH2Cl2 95/Et2O 5 (b). Yellow-orange
solid, 46%; Rf 0.65 (b).
3,4-Dihydroquinoline-2(1H)-thione (21). General procedure for
thionation from 16 (1 g, 6.80 mmol) and Lawesson’s reagent (1
equiv) in dry toluene. Column chromatography with CH2Cl2 (c).
Yellow-orange solid, 77%; Rf 0.36 (c).
N-[3,3-Dimethyl-4-(4-methylaminophenyl)-3,4-dihydroquino-
lin-2-yl]putrescine (7b). General procedure A from 20 (0.200 g,
0.675 mmol, 1 equiv) and putrescine. Column chromatography with
CH3OH/NH4OH 90/10 (j). Oil, 72%; Rf 0.40 (j). HPLC-1 tR 30.03
min, 98.9% pure. Anal. (C22H30N4, 3HCl) C, H, N.
N-(3,4-Dihydroquinolin-2-yl)putrescine (8b). General proce-
dure A from 21 (0.250 g, 1.53 mmol, 1 equiv) and putrescine.
Column chromatography with CH3OH/NH4OH 90/10 (j). Oil, 57%;
Rf 0.21 (j). HPLC-1 tR 24.14 min, 100% pure. Anal. (C13H19N3,
2HCl, 1.2H2O) C, H, N.
(6ar,8â,12br)-N1-(6a,7,8,12b-Tetrahydro-6a,8-dimethyl-9-
methylaminobenzo[k]phenanthridin-6-yl)spermidine (5c). Gen-
eral procedure B from thiolactam 18 (0.4 g, 0.124 mmol, 1 equiv)
and N4,N8-di-(tert-butoxycarbonyl)spermidine. Oil, 65%; Rf 0.29
(isopropylamine/CH3OH/CHCl3 1/4/4, k). HPLC-1 tR 30.66 min,
99.7% pure. Anal. (C27H39N5, 4HCl, 0.9H2O) C, H, N.
N1-(3,3-Dimethyl-4-methylphenylamino-3,4-dihydroquinolin-
2-yl)spermidine (6c). General procedure B from 19 (0.35 g, 1.18
mmol, 1 equiv) and N4,N8-di-(tert-butoxycarbonyl)spermidine. Oil,
40%; Rf 0.36 (k). HPLC-1 tR 30.41 min, 98.4% pure. Anal.
(C25H37N5, 3HCl, 1.3H2O) C, H, N.
N1-[3,3-Dimethyl-4-(4-methylaminophenyl)-3,4-dihydroquino-
lin-2-yl]spermidine (7c). General procedure B from 20 (0.25 g,
0.844 mmol, 1 equiv) and N4,N8-di-(tert-butoxycarbonyl)spermidine.
Oil, 52%; Rf 0.32 (k). HPLC-1 tR 30.45 min, 99.7% pure. Anal.
(C25H37N5, 4HCl, 0.9H2O) H, N; C calcd 52.71, found 53.46.
N1-(3,4-Dihydroquinolin-2-yl)spermidine (8c). General proce-
dure B from 21 (0.2 g, 1.52 mmol, 1 equiv) and N4,N8-di-(tert-
butoxycarbonyl)spermidine. Oil, 25%; Rf 0.23 (k). HPLC-1 tR 28.48
min, 99.5% pure. Anal. (C16H26N4, 3HCl, 1.2H2O) H, N; C calcd
48.26, found 48.92.
(6ar,8r,13ar)-N1-(6a,8,13,13a-Tetrahydro-6a,8,13-trimethyl-
7H-quino[4,3-b][1]benzazepin-6-yl)spermine (4d). General pro-
cedure A from 17 (0.4 g, 1.24 mmol, 1 equiv) and spermine.
Column chromatography with isopropylamine/CH3OH/CHCl3 1/4/4
(k). Oil (50%). Hydrochloride (90%): Rf 0.27 in isopropylamine/
CH3OH/CHCl3 2/4/4 (l). HPLC-1 tR 31.23 min, 100% pure. Anal.
(C30H46N6, 4HCl, 1.2H2O) C, H, N.
(6ar,8â,12br)-N1-(6a,7,8,12b-Tetrahydro-6a,8-dimethyl-9-
methylaminobenzo[k]phenanthridin-6-yl)spermine (5d). General
procedure A from 18 (0.450 g, 1.39 mmol, 1 equiv) and spermine.
Column chromatography with isopropylamine/CH3OH/CHCl3 1/4/4
(k). Oil (31%). Hydrochloride (91%) Rf 0.11 (k), 0.16 (l). HPLC-1
tR 30.59 min, 99.6% pure. Anal. (C30H46N6, 5HCl, H2O) H, N; C
calcd 52.15, found 52.66.
N1-(3,3-Dimethyl-4-methylphenylamino-3,4-dihydroquinolin-
2-yl)spermine (6d). General procedure A from 19 (0.4 g, 1.35
mmol, 1 equiv) and spermine. Column chromatography with
isopropylamine/CH3OH/CHCl3 2/4/4 (l). Oil, 48%; Rf 0.23 (l).
HPLC-1 tR 30.46 min, 100% pure. Anal. (C28H44N6, 4HCl, 2H2O)
C, H, N.
(6ar,8â,13ar)-N-(6a,8,13,13a-Tetrahydro-6a,8,13-trimethyl-
7H-quino[4,3-b][1]benzazepin-6-yl)-3-aminopropan-1-ol (4a).
General procedure A from 17 (0.81 g, 2.4 mmol, 1 equiv) and
3-aminopropan-1-ol. Column chromatography with acetone/NH4-
OH (1%) (d). Oil (34%). Hydrochloride (90%): Rf 0.57 (d), 0.68

242 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Delcros et al.
N1-[3,3-Dimethyl-4-(4-methylaminophenyl)-3,4-dihydroquino-
lin-2-yl]spermine (7d). General procedure A from 20 (0.23 g, 0.777
mmol, 1 equiv) and spermine. Column chromatography with
isopropylamine/CH3OH/CHCl3 2/4/4 (l). Oil, 69%; Rf 0.24 (l).
HPLC-1 tR 30.58 min, 98.8% pure. Anal. (C28H44N6, 5HCl, 0.6H2O)
H, N; C calcd 51.12, found 51.58.
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 in the
culture medium at the time of drug addition. After exposure to the
drug for 48 h, cell growth was determined by measuring formazan
formation from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
by use of a Titertek Multiskan MCC/340 microplate reader
(Labsystems, Cergy-Pontoise, France) for absorbance (540 nm)
measurements.94
Polyamine Uptake Inhibition in L1210 and CHO Cells. The
ability of the amidine derivatives to compete with radiolabeled
putrescine, spermidine, or spermine for uptake was determined in
L1210 cells by a 10 min uptake assay at 37 °C in the presence of
increasing concentrations of competitor, with 9.4 µM [14C]pu-
trescine, 1 µM [14C]spermidine, or 1 µM [14C]spermine as substrate.
The assay mixture contained 2 × 106 L1210 cells and was
performed in a final volume of 0.6 mL of Hanks’ balanced salt
solution supplemented with 20 mM N-(2-hydroxyethyl)piperazine-
N′-2-ethanesulfonic acid (HBSS-HEPES). Initially, the Km values
of putrescine, spermidine, and spermine transport were determined
as previously described.95 Ki values were determined by use of the
Cheng-Prusoff equation96 from the IC50 value derived by iterative
curve fitting of the sigmoidal equation describing the velocity of
polyamine uptake in the presence of the respective competitor.32,97
N1-(3,4-Dihydroquinolin-2-yl)spermine (8d). General procedure
A from 21 (0.23 g, 1.41 mmol, 1 equiv) and spermine. Column
chromatography with isopropylamine/CH3OH/CHCl3 2/4/4 (l). Oil,
59%; Rf 0.11 (l). HPLC-1 tR 29.62 min, 99.1% pure. Anal.
(C19H33N5, 4HCl, 1.3H2O) C, H, N.
Biological Studies. Unless otherwise stated, usual laboratory
chemicals were purchased from Merck (Darmstadt, Germany) or
Sigma (St Louis, MO). DFMO was obtained from Ilex Oncology
(San Antonio, TX). Purified bovine serum oxidase was a generous
gift from Professor B. Mondovi (University La Sapienza, Rome,
Italy). [14C]Putrescine dihydrochloride, [14C]spermidine trihydro-
chloride, [14C]spermine tetrahydrochloride, and [8-3H]adenosine-
3′,5′ cyclic phosphate were obtained from Amersham (Les Ulis,
France). A SPEX Fluorolog 2 spectrofluorometer (Jobin Yvon,
Longjumeau, France) was used for fluorescence measurements. All
data are given as mean values of three or more experiments.
Comparisons between means were made by Student’s t-test, with
the assumption of significance at p < 0.05.
Ethidium Bromide Displacement from Calf Thymus DNA.
The release of ethidium bromide (EB) from the EB-DNA complex
was monitored fluorometrically following the procedure described
by Basu et al.64 EB-DNA complex was formed by adding calf
thymus DNA (0.2 A260 unit) to 3 mL of a solution of EB (2 µM in
50 mM NaCl and 1 mM sodium cacodylate, pH 7.0). The complex
was titrated by adding small volumes of concentrated solutions of
the test compounds at room temperature. Fluorescence ((λexc 546
nm; λem 595 nm) was monitored in 10 mm path length quartz
cuvettes. The fluorescence values were corrected for the dilution
of the complex caused by the addition of compound. CC50 values
were the concentration of compounds that release 50% of the bound
EB.
Assay of Calmodulin-Activated cAMP-Dependent Phos-
phodiesterase. The assay of PDE activity was performed as
previously described.44 Briefly, 0.2 milliunit of PDE (crude
preparation from bovine heart containing calmodulin and Ca2+, or
an activator-deficient PDE from bovine heart) was incubated for
20 min at 37 °C in the presence of 0.1 mM [8-3H]cAMP (10 µCi,
Amersham, Les Ulis, France) and various concentrations of the drug
to be tested in 30 mM Tris-HCl, 3 mM MgSO4, and 0.6 mM AMP.
The enzyme reaction was stopped by adding 50 µL of an aqueous
solution of ZnSO4 (21.5 mM) to the 100 µL reaction mixture. The
addition of 50 µL of a solution of 17.7 mM Ba(OH)2 precipitated
the AMP together with BaSO4. Radioactivity was counted in a 150
µL aliquot of the supernatant.
The ability of the amidine derivatives to inhibit the activity of
the PTS was assayed in CHO cells. CHO cells were grown in 12-
well plates. After three washings in prewarmed PBS, 1 mL of a
prewarmed solution of HBSS-HEPES buffer containing various
concentrations of drug to be tested was added in each well. In some
experiments, 200 µM CHX was added. After incubation at 37 °C
(2-120 min), the medium was eliminated, and the wells were
washed with prewarmed buffer. The reaction was initiated by the
addition of 1 µM [14C]spermidine in 500 µL of buffer. After an
incubation at 37 °C for 5 min, the cells were washed three times
with ice-cold PBS and lysed in 0.1 N NaOH. Aliquots were used
for protein determination98 and for scintillation counting.
Cellular Uptake. For determination of the cellular uptake of
the derivatives, cells were seeded in culture flasks at 4 × 104 cells/
mL for L1210 cells and at 2 × 105 cells/mL for CHO and CHO-
MG cells. Drugs were added at the time of seeding for L1210 and
24 h after seeding for CHO and CHO-MG cells. Cells were
harvested 24 h (CHO and CHO-MG) or 48 h (L1210) after drug
addition. Harvested cells were washed 3 times in cold 0.14 M NaCl.
Cell pellets were disrupted by sonication in 1 mL of 0.2 N HClO4.
After a night at 4 °C, homogenates were centrifuged at 15 000 rpm
for 30 min. Supernatants were used for HPLC determination of
polyamine and derivatives as described above. Pellets were dis-
solved in 0.1 N NaOH and used for protein determination.
Oxidation by Bovine Serum Amine Oxidase. Purified bovine
serum amine oxidase was used for the determination of the substrate
properties of the compounds. Hydrogen peroxide formation was
determined at 10 µM of each compound using the horseradish
peroxidase-catalyzed oxidation of homovanillic acid into a fluo-
rescent product, following a published procedure.93
Cell Culture. Murine leukemia L1210, CHO, and CHO-MG
cells were grown in RPMI 1640 medium supplemented with 10%
fetal calf serum, glutamine (2 mM), penicillin (100 units/mL),
streptomycin (50 µg/mL) (Eurobio, Les Ulis, 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.
Aminoguanidine (2 mM) was added to the culture medium to
prevent the oxidation of the drugs by the serum amine oxidase
present in the calf serum.
In Vitro Evaluation of Drug Cytotoxicity/Cytostasy. The effect
of the amidine derivatives on L1210 cell growth was assayed in
sterile 96-well microtiter plates (Becton Dickinson, Oxnard, CA).
L1210 cells were seeded at 5 × 104 cells/mL of medium (100 µL/
well). Single CHO and CHO-MG cells harvested by trypsinization
were plated at 2 × 103 cells/mL. Drug solutions (5 µL/well) of
Acknowledgment. We are indebted to Professor W. Flintoff
(University of Western Ontario, London, Canada) for providing
the CHO-MG cells and to Professor Mondovi (University La
Sapienza, Rome, Italy) for the gift of purified bovine serum
amine oxidase. We are grateful to D. Moncoq, F. Me´tais, S.
Ginhoux, and R. Havouis for their technical assistance and to
Professor O. Phanstiel for critical reading of the manuscript.
We acknowledge the Centre National de la Recherche Scien-
tifique (CNRS), the Association pour la Recherche sur le Cancer
(ARC), and the Ligue Nationale Contre le Cancer (LNCC,
Commite´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). EU COST 922 is acknowledged for support. Our
sincere thanks are also due to A. Bondon (UMR 6509 CNRS,
Rennes, France) for performing heteronuclear multiple bond
correlation (HMBC) and heteronuclear multiple quantum coher-
ence (HMQC) spectra and Dr P. Guenot (Centre Re´gional de
Mesures Physiques de l’Ouest, Rennes, France) for mass
measurements.

Polyamine Homologation of Heterocyclic Amidines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 243
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