In vitro and in vivo evaluations of THAM derived telomers bearing RGD and Ara-C for tumour neovasculature targeting

Article abstract of DOI:10.1016/S0223-5234(03)00150-8

As an approach to the development of specific drug delivery systems, a new class of low macromolecular carriers called 'telomers' endowed with an antitumour agent, such as arabinofuranosylcytosine (Ara-C), RGDSK peptidic sequences, as tumour targeting moieties, and tyrosine groups labelled with ¹²⁵I atoms allowing the in vivo scintigraphic follow up, were synthesized. Their tumour targeting ability was assessed in vivo in mice bearing a murine B16 melanoma. The biological results showed that the presence of RGDSK sequences onto the macromolecules leads to the selective targeting and the accumulation of telomers within the vascularized zone of the tumour. Moreover, such compounds exhibited in vitro a better IC₅₀ (0.015 μM) than pure Ara-C and in vivo an oncostatic index higher than 160%.

Full text of DOI:10.1016/S0223-5234(03)00150-8

European Journal of Medicinal Chemistry 38 (2003) 825ꢀ836
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Original article
In vitro and in vivo evaluations of THAM derived telomers bearing
RGD and Ara-C for tumour neovasculature targeting
S. Jasseron a, C. Contino-Pe´pin a,*, J.C. Maurizis b, M. Rapp b, B. Pucci a,*
a Laboratoire de chimie bioorganique et des syste`mes mole´culaires vectoriels, Universite´ d’Avignon, 33, rue Louis Pasteur, 84000 Avignon, France
b UMR INSERM 484, rue Montalembert, BP 484, 63005 Clermont Ferrand Cedex, France
Received 13 March 2003; received in revised form 18 July 2003; accepted 29 July 2003
Abstract
As an approach to the development of specific drug delivery systems, a new class of low macromolecular carriers called ‘telomers’
endowed with an antitumour agent, such as arabinofuranosylcytosine (Ara-C), RGDSK peptidic sequences, as tumour targeting
moieties, and tyrosine groups labelled with 125I atoms allowing the in vivo scintigraphic follow up, were synthesized. Their tumour
targeting ability was assessed in vivo in mice bearing a murine B16 melanoma. The biological results showed that the presence of
RGDSK sequences onto the macromolecules leads to the selective targeting and the accumulation of telomers within the
vascularized zone of the tumour. Moreover, such compounds exhibited in vitro a better IC50 (0.015 mM) than pure Ara-C and in
vivo an oncostatic index higher than 160%.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: RGD; Tris; Telomers; Prodrugs; Arabinofuranosylcytosine; Angiogenesis
1. Introduction
targeting of therapeutics to specific tumoural sites could
increase their biological effect and reduce their whole
toxicity.
Currently, a large variety of chemotherapeutic drugs
are widely used to treat malignant diseases. Unfortu-
nately, the remarkable antitumour activity exhibited by
many cytotoxic drugs is accompanied by undesirable
side-effects causing severe damage to healthy tissues and
therefore, limiting their efficacy [1,2]. In this context,
Delivery of cytotoxics to tumour cells is a problematic
challenge because of their genomic instability (i.e. gene
amplification, chromosomal translocations, chromo-
some loss . . .), heterogeneity and rapid mutation leading
to the development of drug resistance [3]. Acquired drug
resistance has been identified as the major cause of
inefficacy of cytotoxic tumour therapy [4]. In contrast,
the endothelial cells lining the tumour vessels consist of
a homogeneous and genetically stable population less
likely to develop resistance to a particular agent [5].
Targeting the tumour vasculature rather than the
tumour cell population itself could also circumvent
one of the major mechanism of drug resistance involving
insufficient drug uptake by the tumour mass.
Abbreviations: AIBN,
a,a?-azobisisobutyronitrile;
Ara-C,
arabinofuranosylcytosine; b-Ala, b-Alanine; BOP, benzotriazol-1-yl-
oxy-tris(dimethylamino)-phosphonium hexafluorophosphate; DCC,
1,3-dicyclohexylcarbodiimide; DCU, 1,3-dicyclohexylurea; DIEA,
diisopropylethylamine;
polymerization; ECM,
DPn, number
extracellular
average
matrix;
degree
Fmoc,
of
9-
fluorenylmethoxycarbonyl; Gaba, g-amino butyric acid; i.v.,
intravenous; p.o., per os; SAR, structure-activity relationship;
TBTU,
tetrafluoroborate;
tris(hydroxymethyl)acrylamidomethane;
O-(1H-benzotriazol-1-yl)-N,N,N?,N?-tetramethyluronium
In fact, a tumour cannot grow beyond the size of 1ꢀ2
/
TEA,
triethylamine;
THAM,
Tris,
mm in diameter without developing a blood supply
providing oxygen and nutrients. This phenomenon,
known as angiogenesis, was hypothesized 30 years ago,
by Judah Folkman, to be an absolute requirement for
the growth and metastasis of solid tumours [6,7].
tris(hydroxymethyl)aminomethane; Mtr, 2,3,6-trimethyl-4-methoxy-
benzenesulfonyl.
* Correspondence and reprints.
E-mail addresses: christine.pepin@univ-avignon.fr (C. Contino-
Pe´pin), bernard.pucci@univ-avignon.fr (B. Pucci).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00150-8

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S. Jasseron et al. / European Journal of Medicinal Chemistry 38 (2003) 825ꢀ836
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If in one hand the easy accessibility of the tumour
mour microvasculature is more leaky to macromolecu-
lar tracers than that of comparable normal tissues [19].
In this context, aside from reducing the toxicity of Ara-
C on normal cells by a selective targeting of angiogenic
sites, we could assume that telomers bearing both Ara-C
and RGD units could concentrate in tumour vessels
easier than free Ara-C.
Work reported herein deals with the synthesis and
preliminary biological assessments of telomeric pro-
drugs of Ara-C allowing the selective targeting of
proliferative angiogenic vascular cells supporting tu-
mours growth.
microenvironment may provide therapeutic opportu-
nities, on the other hand, tumour blood vessels exhibit
several abnormalities in comparison with normal phy-
siological vessels [8] which could also constitute poten-
tial targets for specific antiangiogenic therapy. Indeed,
the vasculature undergoing angiogenesis differs from
normal quiescent vasculature by an overexpression of
ECM-binding integrin receptors such as aVb3 and
aVb5. Other specific markers including adhesion mole-
cules like E-selectin or VEGF receptors are upregulated
on activated endothelial cells, making them suitable
targets for chemotherapeutics administration [9,10].
Many of these phenotypic differences can be exploited
not only as potential therapeutic targets but also to
establish an ‘angiogenic profile’ of patients with cancer
or exhibiting other chronic angiogenic diseases. In this
regard, it is today possible to discriminate newly formed
vessels from those that are more mature using specific
antibodies [11] or by medical imaging techniques based
on nuclear medicine. Several studies indicate that
appropriate integrin activation on endothelial cells is
required for maturation of angiogenic vessels. Intense
researches have been focused on aVb3 integrin and
showed that its blockade during angiogenesis process
results in apoptosis of activated endothelial cells and a
marked decreased of angiogenesis [12]. An example is
the use of specific antagonists of aVb3 receptors such as
monoclonal antibody LM 609 or small cyclic RGD-
containing peptides: these compounds induce rapid
apoptosis only in newly sprouting vessels, without
affecting mature preexisting vessels [13,9].
2. Chemistry
The strong implication of the integrin receptor aVb3
in the development of solid tumours and metastatic
colonies has been widely demonstrated [20,21]. Among
the numerous peptidic ligands of this receptor, the so-
called ‘universal cell recognition sequence’ RGD (Arg-
Gly-Asp) has been one of the most studied [22,23]. This
sequence, known to be a recognition element in many
integrin-dependent cell adhesion processes, is found in
many extracellular matrix proteins like vitronectin,
laminin, fibrinogen and fibronectin [24]. Although this
RGD motif occurs frequently in various proteins, only a
small number of them exhibit an integrin-binding
activity. Indeed, it should be noted that various RGD-
containing peptides, exhibiting a RGD sequence inac-
cessible to integrins, present a limited integrin-binding
activity [25].
This affinity of RGD peptides for aVb3 receptors
expressed on tumour blood vessels and on some
tumoural cells has found major attention. Recently,
this property has been exploited to image tumours in
vitro and in vivo using radiolabelled RGD-containing
peptides [14,15].
All of these interesting data suggested us that an
increased efficiency could be achieved by grafting
simultaneously a cytotoxic agent such as Ara-C and a
RGD-containing oligopeptide onto a carrier. A pioneer-
ing work in this field was previously reported by
Ruoslahti and co-workers [16] who have coupled
doxorubicin with a cyclic RGD peptide.
In an attempt to provide a selective tumour cells
targeting, we chose to introduce RGD moieties on the
low macromolecular drug delivery systems we have been
developing from many years in our team, namely,
‘telomers’ [17]. By this way, we can expect to obtain
telomeric prodrugs of Ara-C exhibiting an increased
affinity for the highly vascularized zone surrounding
tumours. Furthermore, tumour-associated vasculature
is known to be hyperpermeable to macromolecules such
as plasma proteins and other high molecular blood
components [8,18]. Different authors showed that tu-
Many SAR investigations, performed with peptides
selected from random phage display screens [25,26] or
with synthetic linear or cyclic peptides [27], have shown
that the amino acids flanking the recognition motif
influence the affinity of the whole RGD-containing
ligand toward the aVb3 receptor. In particular, hydro-
phobic amino acids following the aspartic acid enhance
this affinity. However, small synthetic RGD-peptides
with serine in position 4 showed a very high inhibitory
activity on the vitronectin-aVb3 interaction. Many
authors suggested that a hydrogen bond between the
hydroxyl group of serine and an acceptor group of the
receptor might be formed in addition to the hydro-
phobic interactions that also take place in this region
[28,29]. With regard to the amino acid in position 5,
neither hydrophobic nor hydrophilic parameters seem to
influence the activity. Considering all these data, and to
provide a good integrins accessibility for the RGD
moieties, we decided to synthesize a peptidic chain
containing a RGDSK sequence. This pentapeptide was
grafted onto the telomer backbone through a g-amino-
butanoic acid spacer arm.
The telomeric carriers, short macromolecules bearing
a functionnalized end-group, are usually obtained by

S. Jasseron et al. / European Journal of Medicinal Chemistry 38 (2003) 825ꢀ
/836
827
free radical copolymerization of acryloyl monomers
derived both from tris(hydroxymethyl)acrylamido-
methane (THAM) and acryloyl peptide in the presence
of an alkane or a perfluoroalkanethiol as a transfer
reagent. In previous publications, we reported that the
physico-chemical parameters of these telomers (mole-
cular weight, Hydrophilic Lipophilic Balance, electric
charge) can be adjusted through both the starting
material and the experimental conditions [30,31]. As
regards their biological properties, we have shown that
these multifunctional carriers are able to cross physio-
logical membranes [32] to diffuse in all tissues. A whole
body autoradiography performed in rat and mouse
allowed us to specify the quite ubiquitous biodistribu-
tion of THAM derived telomers in all biological
compartments except brain [33]. Moreover, after intra-
venous (i.v.) or per os (p.o.) administration to rat, these
compounds were slowly released within 100 h and no
toxicity was observed. Finally, the anchorage of various
antitumour agents such as cytosine arabinoside (Ara-C)
or 5-fluorouracil (5-Fu) to the polymeric backbone gave
macromolecular prodrugs exhibiting, in vitro and in
vivo, an increased bioavailability and a better therapeu-
These four polymerizable monomers 7, 8, 9, 10 gave
satisfactory elemental analysis and were fully character-
ized by NMR spectroscopy.
Telomerization of such monomers, providing amphi-
philic telomers 11a (then 11), 12a (then 12) and 13a
(then 13), was performed as previously described [33,38]
in the presence of alkanethiol as transfer reagent (or
telogen), using AIBN as radical initiator (see Fig. 2).
The AIBN concentration in the reaction mixture was
roughly 10 times lower than the telogen one [39]. The
number average degree of polymerization (DPn) is equal
to the amount of repeating units (xꢁ
/
yꢁ
/
zꢁ
/
w) or (xꢁ
/
yꢁvꢁw). With a given transfer reagent, it may vary
/
/
from one (monoadduct) to several tens, depending on
the (telogen)/(monomers) ratio (R0) adjusted through
both starting material and experimental conditions [30].
The proportions of monomers 7, 8, 9 and/or 10 and
octanethiol used are reported in Table I. These propor-
tions were chosen taking into account previous results
obtained with THAM telomerization [38,31]. Each
experiment was pursued until the complete disappear-
ance of the monomers. Telomers were purified by
chromatography through a Sephadex LH20 column
(for compounds 11a, 12a and 13a) or a Sephadex G50
column and then lyophilized (compounds 11, 12, 13).
The hydrolysis of tert-butyl protective groups of tyr-
osine pendants on compound 11a, by using a trifluor-
tic index than the parent drugs [34ꢀ36].
/
The RGDSK sequence 4 was prepared following
conventional methods of liquid phase synthesis applying
fmoc-strategy (Fig. 1) [37] In order to perform the final
peptide deprotection by using acid hydrolysis, suitable
labile groups were chosen to protect the different amino
acids: t-butyl group for serine and aspartic acid, Mtr
group in the case of arginine. Compounds 1 and 4 were
obtained using respectively BOP and TBTU coupling
reagents in methylene chloride. Pentafluorophenol and
DCC were both used to carry out the synthesis of
synthons 5 and 6. Following this pathway, all com-
pounds were fully characterized by NMR spectroscopy.
The RGDSK protected sequence was prepared with an
overall yield of 43% and gave satisfactory mass spectro-
metric analysis.
In order to follow-up the macromolecules in vivo, a
tyrosine polymerizable monomer 7, easily radiolabelled
with 125I atoms, was used during the preparation of
THAM derived cotelomers 11, 12 and 13. The synthesis
of this monomer 7 started by activating the N-g-
acrylamidobutyric acid as its hydroxysuccinimidyl ester
9. Consecutive coupling of this active ester to O-(tert-
oacetic acidꢀmethylene chloride mixture (3/7 v/v),
/
provided telomer 11 in satisfactory yields. With regard
to the grafting of the peptidic chain to telomers 12a and
13a, the benzyloxycarbonyl protection of the side-chain
amino group of lysine in RGDSK sequence 4 was first
removed by catalytic hydrogenolysis on Pd/C. Conse-
cutive coupling of this peptide 4a to the active ester
groups of telomers 12a and 13a was carried out in
methylene chloride at r.t. for 24 h under a nitrogen
atmosphere. The structure of these cotelomers 11a (or
11), 12a (or 12) and 13a (or 13), i.e. the relative
proportions of each tyrosine (x), peracetylated THAM
(y), active ester or RGD (z or v) and/or Ara-C (w)
moiety in the cotelomer and the DPn of the macro-
1
molecule, were determined in H-NMR by comparing
the area of typical signals of each monomer.
For example, in the case of telomer 11, x, y and w
values were determined by comparing peaks area
assigned to the terminal methyl signal in the hydro-
carbon tail (d 0.9 ppm, integral 3H), respectively to
aromatic tyrosine protons (d 6.89 ppm, integral 2xH),
to methylene protons of peracetylated THAM (d 4.32
ppm, integral 6yH) and to H6 proton of Ara-C moieties
(d 8.14 ppm, integral 1wH). The respective value of z
for telomers 12a and 13a, was determined as above, by
comparing peaks area assigned to the terminal methyl
signal in the hydrocarbon tail (d 0.9 ppm, integral 3H)
to succinimidyl protons of active ester groups (d 2.86
ppm, integral 4zH).
butyl) tert-butyl L tyrosinate was performed in methyl-
ene chloride at room temperature (r.t.) and provided
monomer 7 in satisfactory yield (87%).
Peracetylated THAM monomer 8 was easily synthe-
sized from THAM using the conventional reactant
acetic anhydride in pyridine at r.t. for 12 h. Compound
8 was obtained after recrystallization in an ethyl
acetateꢀhexane mixture as a white powder (87% yield).
/
The preparation of the Ara-C polymerizable monomer
10 was described in a previous paper [35].

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S. Jasseron et al. / European Journal of Medicinal Chemistry 38 (2003) 825ꢀ836
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Fig. 1. Synthesis of the RGDSK sequence [37].
The proportion of RGD sequences grafted onto
telomers 12 and 13 was specified, before the deprotec-
the molecules with 125I. Using these labelled com-
pounds, their biodisposition was studied in mice bearing
syngenic B16 melanoma tumours. The pharmacological
activity of the molecules 11 and 13 bearing Ara-C was
studied on B16 melanoma in vitro, using the measure of
the coloning efficiency (colony forming) and in vivo, by
determining the oncostatic index after treatment by Ara-
C equivalent doses of 11 and 13 on mice bearing grafted
B16 melanoma.
1
tion of Mtr groups, by comparing in H-NMR peaks
area assigned to the aromatic Mtr proton (d 6.56 ppm,
1vH) to the terminal methyl signal in the hydrocarbon
tail. It is noteworthy that for each telomer, the NMR
structural evaluation showed that the proportion of
RGD moieties was similar to the succinimidyl groups
one in the precursor telomers 12a and 13a. Finally, the
hydrolysis of both tert-butyl and Mtr protective groups,
using a trifluoroacetic acidꢀ
/
methylene chlorideꢀmethyl
/
phenyl sulfide mixture (3/5/2 v/v/v) at r.t. for 24 h, was
4. Results and discussion
1
checked by H-NMR spectrum after a purification step
and gave compounds 12 and 13 in good conditions.
Tumour growth and metastasis have been proved to
be dependent on the development of new capillaries
from preexisting blood vessels, namely ‘angiogenesis’.
Many authors reported that angiogenesis depends on
specific endothelial cell adhesive events mediated by
integrin aVb3 [40,41] which presents a high affinity for
RGD peptides.
3. Pharmacology
A tyrosine residue was introduced in the chemical
structure of telomers in order to perform a labelling of

S. Jasseron et al. / European Journal of Medicinal Chemistry 38 (2003) 825ꢀ
/836
829
Fig. 2. Synthesis of telomeric carriers derived from THAM.
In this context, we expected that the introduction of
RGD pendants on our macromolecular carriers called
‘telomers’ could be a convenient approach for the
selective delivery of chemotherapeutics to tumoural
zones.
A series of three amphiphilic telomers radiolabelled
by 125I was then synthesized. Different structural para-
meters likely to enhance the potency of the prodrug
towards tumoural tissues have been considered and their
influence evaluated. Among them, for roughly similar

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S. Jasseron et al. / European Journal of Medicinal Chemistry 38 (2003) 825ꢀ836
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Table I
Experimental data of the cotelomers synthesis
Initial Conditions [monomer]/[telogen]
Solvent Telomer structures
Compounds
7
8
9
10
x
y
z
w
DPn Mw
Yield (%)
Compound/final Mw
11a
12a
13a
5
8
5
15
14
20
0
6
10
0
DMF
THF
DMF
3.5 23
15 11
2.5 16
0
5.5 32 10 700 73
11/10 300
12/14 100
13/10 800
5
0
2
31
26.5
9600 75
8300 54
10
10
6
x, y, z, and w are the average numbers derived from tyrosine (x), peracetylated THAM (y), active ester or RGD (z) and Ara-C (w) moiety in the
cotelomers.DPnꢂnumber average degree of polymerization equal to xꢁyꢁzꢁw.
/
/
/
/
telomeric sizes (i.e. their DPn), we studied the effect of
the number of RGD pendants and the multiplicity of
Ara-C moieties on the macromolecular backbone.
All compounds were well soluble in water and so,
were likely to be administered to mice by i.v. injection.
Their biodistribution was specified by measuring the
radioactivity levels in several tissues as a function of
time following administration.
Photograph 1 and results reported in Table II clearly
show the significant affinity of telomers 12 and 13,
bearing RGDSK sequences, for the vascularized zone of
the tumour while telomer 11 does not show any affinity.
Indeed, for these compounds, the ratio stroma/blood is,
whatever the time, always higher than 1. Comparatively,
telomer 11, without any RGDSK moieties, does not
concentrate in these compartments and so exhibits no
affinity for neither angiogenic nor tumoural sites (Table
II). This lack of affinity of telomer 11 for specific
compartments of the body is in good agreement with the
homogeneous biodistribution of radiolabelled telomers
derived from THAM previously observed [33].
Moreover, if we consider the relative proportion of
telomers 12 and 13 in each tumoural and stroma zone,
we can note an increase of the ratio tumour/stroma
versus time, underlining the diffusion of these prodrugs
toward the tumour mass. However, 6 h after adminis-
tration, tumoural tissue exhibits slightly lower concen-
tration values for 12 than for 13, while blood
concentration decrease is higher for 13, probably linked
to a faster urinary elimination, as confirmed by the
significantly higher kidney radioactivity for 13 at this
time. Such an elimination rate could be due to the
presence of Ara-C moieties on the telomer backbone
which increase its hydrophilic character.
These biological observations demonstrate the key
role of RGD moieties for the specific targeting of highly
vascularized zones surrounding the tumour. However,
by comparing the behaviour of compounds 12 and 13
which were endowed with different RGD sequences
number, no cluster effect could be noted. The affinity of
these telomers for the vascularized zone cannot be really
correlated to the number of RGD groups they own.
The in vitro and in vivo antitumour potencies of the
two telomers 11 (without RGDSK sequences) and 13
bearing both Ara-C were investigated on B16 melanoma
by measuring the coloning efficiency and the oncostatic
index. Fig. 3 shows the dose-effect curves for com-
pounds 11 and 13. From these results, we determined an
IC50 of 0.015 mM for 11 and 0.26 mM for 13,
(comparatively, telomer 12; without Ara-C units exhi-
bits no cytotoxic activity). Oncostatic indexes were 162%
for 11 and 153% for 13. These results are in accordance
with those observed for biodistribution study. Indeed,
although 11 was more than 10 fold efficient than 13 for
the coloning efficiency test, its oncostatic index was only
sligthly superior to this of 13. This discrepancy between
the in vitro and in vivo tests can be explained by a higher
concentration of 13 in the tumoural tissue due to the
binding of RGD residues to angiogenic zones.
In conclusion, all of these biological evaluations
validate the potentialities of THAM derived telomers
endowed with specific oligopeptidic sequences such as
RGD for targeting chemotherapeutics to the activated
endothelium supporting the growth of the tumours. The
study of their therapeutic activity is currently underway
on mice bearing B16 syngenic melanoma and will be
reported in a forthcoming publication.
5. Experimental protocols
The progress of the reactions and the homogeneity of
the compounds were monitored by thin-layer chroma-
Plate 1. Mouse section 1 h after injection of telomer 13 (bearing
RGDSK sequences).

S. Jasseron et al. / European Journal of Medicinal Chemistry 38 (2003) 825ꢀ
/836
831
Table II
Distribution of radioactivity in tissues after i.v. administration of 20 mCi of labelled telomers 11, 12, 13.
Time after administration
15 mn
1 h
6 h
Compounds
11
12
13
11
12
13
11
12
13
Tumour
Stroma
Blood
3.89
4.29
4.99
ND
6.89
2.99
7.59
/
0.6
0.7
1.1
1.749
6.349
3.299
3.369
31.49
2.119
8.579
/
0.65
0.65
0.28
0.56
3.349
6.6291.55 1.19
5.5291.09 3.49
3.6490.58 ND
25.7295.05 4.79
3.0290.57 2.19
21.589
/
0.88 0.99
/
0.2
0.2
0.7
2.149
4.569
1.999
1.489
18.59
0.949
54.5910.2
/
0.59
0.90
0.22
0.20
1.579
3.849
1.569
1.459
32.809
1.3390.54 1.59
65.569
/
0.17 ND
1.449
1.7690.24 2.029
1.0990.08 0.659
0.9590.11 ND
8.0191.54 13.809
0.5290.12 0.4590.2
75.4915.2 85.82914.12
/
0.27 2.029
/
0.42
0.42
0.11
/
/
/
/
/
/0.17 ND
/
/
/
/
/
/
/
/0.24 1.29
/
0.3
/
/
Skin
/
/
/
/0.32 ND
/
Kidney
Liver
/
0.4
0.3
1.2
/
5.26
/
/
0.5
0.4
/
3.19
/2.98 2.29
/0.3
/
/
3.04
/
/
0.35
1.15
/
/
/0.17
/
/0.4
/
/
Thyroid
Stroma/blood
Tumour/stroma
/
/
/
2.65 10.79
/
2.1
/
/9.27 14.49
/3.7
/
/
0.85
0.90
1.92
0.27
1.20
0.50
0.32
0.81
2.29
0.47
2.46
0.40
ND
ND
1.61
0.82
3.10
1
Results, given in percentage of administered doses per g of tissue, are the mean of five experiments9
/
SD.
tography (TLC, Merck F254). Detection was achieved by
either exposure of plates to UV light (254 nm) or by
rotations were measured with a PerkinꢀElmer Model
/
241 polarimeter. Elemental analysis were conducted by
the ‘Service Central de Microanalyse du CNRS’ at Lyon
(France). Analyses indicated by the symbols of the
charring with a methanolꢀsulfuric acid (1:1) solution.
/
Purifications were performed by column chromatogra-
phy over silica gel (Merck 60), or by gel-permeation on
Sephadex G50 or LH20 (Pharmacia LKB). Melting
points (m.p.) were measured on an electrothermal 9100
elements or functions were within 9
/
0.4% of theoretical
values.
Reactions were performed in anhydrous conditions
under dry nitrogen. All the solvents were distilled and
dried according to standard procedures. For telomeriza-
tion step, the solutions were carefully deoxygenated by
nitrogen bubbling before use. AIBN was purified twice
by recrystallization from absolute ethanol.
1
type-apparatus and are reported uncorrected. The H-,
13C- and 19F-nuclear magnetic resonance (NMR) spec-
tra were recorded at 250 MHz on a Bruker AC250
spectrometer in chloroform-d3 (CDCl3), methanol-d4
(CD3OD) or dimethyl sulfoxyde-d6 (DMSO). The
chemical shifts are expressed in parts per million
(ppm) relative to tetramethylsilane as an internal
reference for the 1H and 13C spectra. For the 19F-
NMR spectra, the internal reference is CFCl3. Optical
THAM, Ng-acrylamidobutyric acid and N-(3-(N4-
carbamoyl-1-b-D-arabino-furanosylcytosine)-propyl ac-
rylamide (compound 10) were synthesized as described
by Pucci et al. [31,35].
Fig. 3. Coloning efficiency measured on B16 melanoma cells as a function of 11 and 13 concentrations. Each value is the mean of four experiments9
/
SD.

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S. Jasseron et al. / European Journal of Medicinal Chemistry 38 (2003) 825ꢀ836
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6. Synthesis of RGDSK sequence [37]
1H, Hb Ser), 3.74 (s, 3H, CH3 methyl ester Lys), 3.41
(dd, 1H, Hb Ser), 3.17 (m, 2H, 2Ho Lys), 2.88 (dd, 1H,
Hb Asp), 2.72 (dd, 1H, Hb Asp), 1.48 (s, 9H, 3 CH3 tert-
butyl ester Asp), 1.84-1.28 (m, 6H, 2Hb, 2Hg, 2Hd Lys),
1.22 (s, 9H, 3 CH3 tert-butyl ether Ser); 13C-NMR
(CDCl3): d 172.96, 171.74, 171.14 and 170.52 (CO Lys,
CO Ser, 2 CO Asp), 157.06 and 156.65 (2 CO urethane
Fmoc and Z), 144.32, 141.94, 137.28, 129.14, 128.71,
128.41, 127.76, 125.72 and 120.65 (C arom. Fmoc and
Z), 82.59-28.67 (tert-butyl ester Asp), 74.80, 27.97 (tert-
butyl ether Ser), 68.04 and 67.88 (CH2 Fmoc and CH2
benzyl Z), 61.61 (Cb Ser), 53.99 (Ca Ser), 52.95 and 52.70
(C methyl ester Lys and Ca Lys), 52.04 (Ca Asp), 47.74
(CH Fmoc), 41.32 (Co Lys), 38.25 (Cb Asp), 32.55 (Cd
Lys), 29.93 (Cb Lys), 22.99 (Cg Lys); MS (FAB): m/z
6.1. Fmoc-Ser(tBu)-Lys(Z)-OMe (1)
To a solution of Clꢃ ꢁH3N-Lys(Z)-OMe (0.99 g, 3
mmol) and DIEA (0.39 g, 3 mmol) in CH2Cl2 (10 mL)
were successively added Fmoc-Ser(tBu)-OH (1.15 g, 3
mmol) and BOP (1.72 g, 3,9 mmol). The pH was
maintained between 8 and 9 and the reaction mixture
left at r.t. overnight. The organic phase was then washed
with 1 N HCl (2ꢄ
/
10 mL), saturated aq. NaHCO3 (2ꢄ
/
10 mL) and water (2ꢄ/10 mL). Drying on Na2SO4 and
removal the solvent in vacuo afforded a white powder
which was used without further purification (1.93 g,
98%): m.p. 60.1 8C (dec.); [a]D
ꢁ
17.9 (c 1, CH2Cl2); 1H-
/
NMR (CDCl3): d 7.75 (d, 2H, arom. Fmoc), 7.60 (d,
2H, arom. Fmoc), 7.43-7.21 (m, 5H, arom. Fmoc, NH
Lys), 7.31 (s, 5H, arom. benzyl Lys), 5.78 (d, 1H, NH
Ser), 5.08 (s, 2H, CH2 benzyl Lys), 4.84 (t, 1H, NH Z
Lys), 4.60 (m, 1H, Ha Lys), 4.39 (d, 2H, CH2 Fmoc),
4.24 (m, 2H, Ha Ser, CH Fmoc), 3.81 (dd, 1H, Hb Ser),
3.73 (s, 3H, CH3 methyl ester Lys), 3.39 (dd, 1H, Hb
Ser), 3.17 (m, 2H, 2Ho Lys), 1.85-1.29 (m, 6H, 2Hb Lys,
2Hg Lys, 2Hd, Lys), 1.22 (s, 9H, 3 CH3 tert-butyl ether
Ser); 13C-NMR (CDCl3): d 172.94 (CO Lys), 170.88
(CO Ser), 157.08 and 156.67 (2 CO urethane Fmoc and
Z), 144.52, 144.36, 141.93, 137.23, 129.13, 128.71,
128.35, 127.71, 125.76 and 120.62 (C arom. Fmoc and
Z), 74.96, 27.99 (tert-butyl ether Ser), 67.80 and 67.24
(CH2 Fmoc and CH2 Z), 62.39 (Cb Ser), 54.93 (Ca Ser),
52.99 and 52.74 (C methyl ester Lys and Ca Lys), 47.76
(CH Fmoc), 41.27 (Co Lys), 32.74 (Cd Lys), 30.02 (Cb
Lys), 22.92 (Cg Lys).
831 [Mꢁ
/
H]ꢁ, 853 [MꢁNa]ꢁ.
/
6.3. Fmoc-Gly-Asp(tBu)-Ser(tBu)-Lys(Z)-OMe (3)
To a solution of the tripeptide resulting from Fmoc
deprotection of 2 (0.5 g, 0.6 mmol) in 10 mL of
piperidineꢀ/methylene chloride (1:9, v/v), was added
active ester 6 (0.28 g, 0.6 mmol) and DIEA (pH 8ꢀ
/9)
in CH2Cl2 (10 mL). The stirring was pursued for 16 h at
r.t. The solution was then evaporated under reduced
pressure and the crude product was purified by chro-
matography (EtOAcꢀhexane, 60:40, v/v) to give a white
/
powder 3 (0.43 g, 80%); m.p. 53.3 8C (dec.); [a]2D0
ꢁ4.6 (c
/
1
1, CH2Cl2); H-NMR (CDCl3): d 7.76 (d, 2H, arom.
Fmoc), 7.60 (d, 2H, arom. Fmoc), 7.45-7.22 (m, 7H,
arom. Fmoc, NH Lys, NH Ser, NH Asp,), 7.34 (s, 5H,
arom. benzyl Lys), 5.83 (d, 1H, NH Gly), 5.14 (t, 1H,
NH Z Lys), 5.07 (s, 2H, CH2 benzyl Lys), 4.82 (m, 1H,
Ha Asp), 4.57 (m, 1H, Ha Lys), 4.45-4.35 (m, 3H, Ha
Ser, CH2 Fmoc), 4.20 (t, 1H, CH Fmoc), 3.89 (m, 2H,
2Ha Gly), 3.75 (dd, 1H, Hb Ser), 3.68 (s, 3H, CH3
methyl ester Lys), 3.39 (dd, 1H, Hb Ser), 3.15 (m, 2H,
2Ho Lys), 2.88 (dd, 1H, Hb Asp), 2.68 (dd, 1H, Hb Asp),
1.41 (s, 9H, 3 CH3 tert-butyl ester Asp), 1.91-1.29 (m,
4H, 2Hb, 2Hg Lys), 1.22 (s, 9H, 3 CH3 tert-butyl ester
Ser); 13C-NMR (CDCl3): d 173.00, 171.90, 170.75,
170.49, 169.76 (CO Lys, CO Ser, 2 CO Asp, CO Gly),
157.31 and 157.13 (2 CO urethane Fmoc and Z), 144.45,
141.98, 137.27, 129.19, 128.78, 128.42, 127.78, 125.77,
120.68 (C arom. Fmoc and Z), 82.78, 28.67 (tert-butyl
ester Asp), 74.78, 28.00 (tert-butyl ether Ser), 68.05 and
67.30 (CH2 Fmoc and CH2 benzyl Z), 61.60 (Cb Ser),
54.19 (Ca Ser), 53.01 and 52.72 (C methyl ester Lys and
Ca Lys), 50.28 (Ca Asp), 47.76 (CH Fmoc), 45.26 (Ca
Gly), 41.38 (Co Lys), 37.68 (Cb Asp), 32.58 (Cd Lys),
29.99 (Cb Lys), 23.03 (Cg Lys); MS (FAB): m/z 888
6.2. Fmoc-Asp(tBu)-Ser(tBu)-Lys(Z)-OMe (2)
Dipeptide 1 (1.3 g, 1.97 mmol) has been treated with
piperidine (10% in CH2Cl2) (10 mL) for 1 h at r.t. The
organic phase was then washed with 1 N HCl (2ꢄ
mL) or until pH 1, saturated aq. NaHCO3 (2ꢄ10 mL)
and water (2ꢄ10 mL). Drying on Na2SO4, the solvent
/
10
/
/
was evaporated under vacuum. To a stirred mixture of
the resulting amine in CH2Cl2 (10 mL) were added
DIEA (pH 8), active ester 6 (1.14 g, 1.97 mmol) and the
mixture was stirred overnight at 25 8C. The solution was
then concentrated under reduce pressure. The residue
was purified by column chromatography on silica gel
using ethyl acetateꢀ
provide the pure product 2 as a white powder (0.97 g,
8.6 (c 1, CH2Cl2);
/
hexane (50:50 v/v) as eluent to
59.2%); m.p. 68.4 8C (dec.); [a]2D0
ꢁ
/
1H-NMR (CDCl3): d 7.78 (d, 2H, arom. Fmoc), 7.61 (d,
2H, arom. Fmoc), 7.46-7.23 (m, 6H, arom. Fmoc, NH
Lys, NH Ser), 7.36 (s, 5H, H arom. benzyl Lys), 5.93 (d,
1H, NH Asp), 5.11 (s, 2H, CH2 Z Lys), 4.91 (t, 1H, NH
Z Lys), 4.57 (m, 2H, Ha Asp, Ca Lys), 4.48-4.42 (m, 3H,
Ha Ser, CH2 Fmoc), 4.26 (t, 1H, CH Fmoc), 3.85 (dd,
[Mꢁ
/
H]ꢁ, 910 [MꢁNa]ꢁ.
/

S. Jasseron et al. / European Journal of Medicinal Chemistry 38 (2003) 825ꢀ
/836
833
6.4. Boc-Arg(Mtr)-Gly-Asp(tBu)-Ser(tBu)-Lys(Z)-
OMe (4)
(dd, 1H, Hb Asp), 2.90 (dd, 1H, Hb Asp), 1.48 (s, 9H, 3
CH3 tert-butyl ester Asp); 13C-NMR (CDCl3): d 170.35
and 168.09 (2 CO ester), 156.55 (CO urethane Fmoc),
144.43, 144.26, 142.01, 128.47, 127.78, 125.79, 120.71 (C
arom. Fmoc), 143.82-136.34 (CF), 83.39-28.68 (tert-
butyl ester Asp), 68.21 (CH2 Fmoc), 50.97 (Ca Asp),
47.76 (CH Fmoc), 38.31 (Cb Asp); 19F-NMR (CDCl3):
The reaction was carried out using tetrapeptide 3 (0.4
g, 0.45 mmol), which was deprotected in 10 mL of
piperidineꢀ
OH (0.22 g, 0.45 mmol), TBTU (0.19 g, 0.58 mmol) and
DIEA until pH 8ꢀ9 in CH2Cl2 (10 mL). Solvent was
/methylene chloride (1:9, v/v), Boc-Arg(Mtr)-
/
d ꢃ
/
152.22 (2F), ꢃ
/
157.36 (1F), ꢃ162.10 (2F).
/
evaporated under vacuum and the crude product
purified by column chromatography on silica gel using
6.6. Fmoc-Gly-OPFP (6)
ethyl acetateꢀ
/
hexane (90:10, v/v) as eluent, to provide
the pure product 4 as a white powder (0.42 g, 92.5 %):
This compound was prepared from Fmoc-Gly-OH
and pentafluorophenol similarly to 5. Yield 61.3%; m.p.
1
2.9 (c 1, CH2Cl2); H-NMR
m.p. 80.2 8C (dec.); [a]2D0
ꢃ
/
(CDCl3): d 7.64 (d, 1H, NH Asp), 7.53 (d, 1H, NH Gly),
7.49-7.29 (m, 7H, arom. Z Lys, NH Lys, NH Ser), 6.54
(s, 1H, H arom. Mtr), 6.34-6.15 (m, 3H, 3 NH guanidine
Arg), 5.65 (d, 1H, NH Boc Arg), 5.24 (t, 1H, NH Z Lys),
5.10 (s, 2H, CH2 Z Lys), 4.77 (m, 1H, Ha Asp), 4.46 (m,
2H, Ha Lys, Ha Ser), 4.22 (m, 1H, Ha Arg), 3.89-3.70
(m, 3H, 2Ha Gly, Hb Ser), 3.84 (s, 3H, CH3 ether Mtr
Arg), 3.71 (s, 3H, CH3 methyl ester Lys), 3.50 (dd, 1H,
Hb Ser), 3.21 (m, 2H, 2Hd Arg), 3.17 (m, 2H, 2Ho Lys),
2.90-2.64 (m, 8H, 2 CH3 Mtr Arg, 2Hb Asp), 2.15 (s, 3H,
CH3 Mtr Arg), 1.84-1.19 (m, 37H, 9 CH3 tert-butyl ester
Asp, tert-butyl ether Ser, tert-butyl Boc Arg, 2Hb, 2Hg,
2Hd Lys, 2Hb, 2Hg Arg); 13C-NMR (CDCl3): d 174.32,
174.13, 172.85, 171.48, 171.35, 170.92, 170.18 (CO Lys,
CO Ser, 2 CO Asp, CO Gly, CO Arg, C guanidin Arg),
158.96, 157.20 and 156.55 (2 CO urethane Boc and Z,
156.3ꢀ
/
157.4 8C; [a]D20
ꢃ
1.3 (c 1, CH2Cl2); 1H-NMR
/
(CDCl3): d 7.82 (d, 2H, arom. Fmoc), 7.65 (d, 2H,
arom. Fmoc), 7.47-7.25 (m, 4H, arom. Fmoc), 5.40 (t,
1H, NH), 4.54-4.42 (m, 5H, CH2 Fmoc, CH Fmoc, 2Ha
Gly); 13C-NMR (CDCl3): d 167.14 (CO ester Gly),
156.84 (CO urethane), 144.31, 142.00, 128.46, 127.77,
125.66, 120.70 (C arom. Fmoc), 68.14 (CH2 Fmoc),
47.73 (CH Fmoc), 42.87 (Ca Gly); 19F-NMR (CDCl3) d
ꢃ
/
152.44 (2F), ꢃ
/
157.25 (1F), ꢃ161.96 (2F).
/
7. Synthesis of monomers
7.1. N-(Ng-Acrylamido)butyramido-tbutyl-(tBu)-
tyrosinyl ester (7)
CÃ
/
OCH3 arom. Mtr Arg), 139.07, 137.25, 137.06,
To a stirred mixture of Tyr(tBu)-tBu (0.065 g, 0.2
mmol) in CH2Cl2 (5 mL) were added DIEA to obtain
pH 8, N?-acrylamido-hydroxysuccinimidyl-butyric ester
9 (0.05 g, 0.2 mmol), HOBT (0.003 g, 0.02 mmol) and
stirred overnight at 25 8C. The solution was then
concentrated under reduce pressure and the residue
purified by column chromatography on silica gel using
134.19, 129.07, 128.87, 128.61, 125.30, 112.28 (C arom.
Mtr and Z), 82.37 (C tert-butyl ester Asp), 80.53 (C tert-
butyl Boc Arg), 74.63 (C tert-butyl ether Ser), 67.09
(CH2 benzyl Lys), 60.96 (Cb Ser), 55.99 (C methyl ether
Mtr Arg), 54.57 and 54.23 (Ca Ser, Ca Arg), 52.87 and
52.74 (C methyl ester Lys and Ca Lys), 50.37 (Ca Asp),
43.92 (Ca Gly), 41.25 and 40.75 (Co Lys, Cd Arg), 37.59
(Cb Asp), 32.27 (Cd Lys), 30.33 and 29.83 (Cb Lys and
Cb Arg), 28.94, 28.56, 27.86 (9 C methyl tert-butyl Asp,
Ser and Arg), 25.93 (Cg Arg), 24.74 (Cg Lys), 23.06,
18.95, 12.52 (3 C methyl Mtr Arg); MS (FAB): m/z 1135
ethyl acetate as eluent to give 7 (0.074 g, 87%); m.p.
115.7 8C; [a]D20
1
38.9 (c 1, CH2Cl2); H-NMR
114.8ꢀ
/
ꢁ
/
(CDCl3): d 7.10 (d, 2H, arom. Tyr), 6.91 (m, 3H, arom.
Tyr, NH Tyr), 6.71 (d, 1H, NH), 6.26-6.16 (m, 2H, 1H
vinylic CH2, vinylic CH), 5.63 (dd, 1H, 1H vinylic CH2),
4.71 (m, 1H, Ha Tyr), 3.34 (dd, 2H, 2Hb), 3.04 (m, 2H,
[Mꢁ
/
H]ꢁ, 1157 [MꢁNa]ꢁ.
/
CH2Ã
/
NH), 2.27 (t, 2H, CH2Ã
/
CO), 1.84 (m, 2H, CH2);
6.5. Fmoc-Asp(tBu)-OPFP (5)
13C-NMR (CDCl3): d 172.99, 171.5, 166.55 (3 CO),
154.97 (Cb Tyr), 131.72, 131.70, 130.50, 126.75, 124.75
(C arom. Tyr, vinylic CH2, vinylic CH), 82.95, 79.07,
29.5, 28.61 (2 tert-butyl Tyr), 54.40 (Ca Tyr), 38.11
Pentafluorophenol (0.68 g, 3.66 mmol) was added to a
solution of Fmoc-Asp(tBu)-OH (1.36 g, 3.06 mmol) and
DCC (0.76 g, 3.66 mmol) in methylene chloride. The
mixture was stirred for 15 h at r.t. and the precipitated
DCU was filtered. The filtrate was evaporated under
vacuum and the residue crystallized with ethyl acetate/
(CH2Ã
m/z 433 [Mꢁ
[2Mꢁ
Na]ꢁ; Anal. C24H36N2O5 (C, H, N).
/
NH), 34.50 (CH2Ã
/
CO), 25.62 (CH2); MS (FAB):
/
H]ꢁ, 455 [Mꢁ
/
Na]ꢁ, 865 [2MꢁH]ꢁ, 889
/
/
heptane to give 5 (1.52 g, 86.2%); m.p. 94.6ꢀ
/
95.8 8C;
2.3 (c 1, CH2Cl2); H-NMR (CDCl3): d 7.77 (d,
7.2. Tris(acetoxymethyl)-acrylamidomethane (8)
1
[a]D20
ꢃ
/
2H, arom. Fmoc), 7.61 (d, 2H, arom. Fmoc), 7.43-7.28
(m, 4H, arom. Fmoc), 5.98 (d, 1H, NH), 4.98 (m, 1H,
Ha Asp), 4.49-4.23 (m, 3H, CH2 Fmoc, CH Fmoc), 3.15
Tris(hydroxymethyl) acrylamido methane (5 g, 28.6
acetic anhydride
(50:50, v/v) (100 mL) at 0 8C. The stirring was pursued
mmol) was added slowly in pyridineꢀ
/

834
S. Jasseron et al. / European Journal of Medicinal Chemistry 38 (2003) 825ꢀ836
/
overnight at r.t. The organic phase was then washed
with 1 N HCl (3ꢄ10 mL), satured aq. NaHCO3 (2ꢄ10
mL) and water (2ꢄ10 mL). After drying on Na2SO4
charring with a methanolꢀsulfuric acid solution) were
/
/
/
evaporated under reduce pressure to give 11a, 12a, 13a.
The DPn was specified by 1H-NMR in CD3OD by
comparing the area of the signal due to the terminal
methyl group of the telomer (distinct triplet; d 0.9) with
the area of the signal ascribed to aromatic protons of 7
(doublet; d 6.9), to methylene protons of 8 (singlet; d
4.3), to succinimidyl protons of 9 (singlet; d 2.9) or to
aromatic proton of 10 (doublet; d 8.1).
/
and removal the solvent in vacuo, the residue crystal-
lized in ethyl acetate/heptane (7/3 v/v) to give 8 (7.52 g,
1
87.3%); m.p. 96.5ꢀ
/
97.4 8C; H-NMR (CDCl3): d 6.34-
6.06 (m, 3H, 1H vinylic CH2, vinylic CH, NH), 5.72 (d,
1H, vinylic CH), 4.53 (s, 6H, 3 CH2), 2.14 (s, 9H, 3
CH3); 13C-NMR (CDCl3): d 171.34 and 166.14 (CO
ester and CO amide), 131.47 (vinylic CH), 127.85
(vinylic CH2), 63.43 (3 CH2), 59.00 (C), 21.42 (CH3);
Anal. C13H19NO7 (C, H, N).
8.2. Deprotection of cotelomer 11a
0.1 g of 11a was stirred 24 h in 10 mL of a
7.3. Ng-Acrylamido-hydroxysuccinimidyl-butyric ester
(9)
trifluoroacetic acidꢀmethylene chloride mixture (3:7, v/
/
v). The solution was then concentrated under vacuum
until TFA was evaporated and poured into diethyl
ether. The precipitate obtained was filtered, thoroughly
washed with diethyl ether and dissolved in a few mL of
water. The mixture was fractionated with a Sephadex
Hydroxysuccinimide (0.73 g, 6.37 mmol) was added
to a solution of Ng-acrylamidobutyric acid (1 g, 6.37
mmol) and DCC (1.57 g, 7.64 mmol) in methylene
chloride. The mixture was stirred for 15 h at r.t. and the
precipitated DCU was filtered. The filtrate was concen-
trated under reduce pressure and the residue purified by
G50 column (2.5ꢄ
fractions (detected on TLC by charring with
methanolꢀsulfuric acid solution) were lyophilized to
/80 cm, eluent: H2O) and the desired
a
/
column chromatography on silica gel (eluent: EtOAcꢀ
/
give a set of telomers 11 as a white powder (0.079 g,
82%).
hexane, 90:10, v/v) to give 9 (0.76 g, 47%): m.p. 93.5ꢀ
/
1
95.4 8C; H-NMR (CDCl3): d 6.29 (m, 2H, vinylic H
and NH), 6.12 (dd, 1H, vinylic CH2), 5.64 (dd, 1H,
vinylic CH2), 3.45 (q, 2H, 2Ha), 2.86 (s, 4H, 4H
succinimidyl), 2.68 (m, 2H, 2Hb), 2.05 (t, 2H, 2Hg);
13C-NMR (CDCl3): d 193.00 (CO ester), 191.83; 188.89
8.3. RGDSK coupling (to 12a and 13a)
Example: coupling of RGDSK to 12a.
After deprotection of the benzyloxycarbonyl group of
compound 4 (0.143 g, 0.126 mmol) by catalytic hydro-
genolysis on Pd/C, the resulting deprotected peptide 4a
(0.126 g, 0.126 mmol) was added to a solution of telomer
12a in methylene chloride (0.1 g, 0.0105 mmol) and the
(3 COÃ
29.34 (2 C succinimidyl), 17.29 (Ca), 13.4 (Cg), 12.00
(Cb); MS (FAB): m/z 255 [Mꢁ
H]ꢁ, 277 [MꢁNa]ꢁ,
509 [2Mꢁ
H]ꢁ, 531 [2MꢁNa]ꢁ; Anal. C11H14N2O5 (C,
H, N).
/N), 144.72 (vinylic CH2), 139.65 (vinylic CH),
/
/
/
/
mixture was stirred 24 h in the presence of DIEA (pH 8ꢀ
/
9). The mixture was concentrated under reduce pressure
and the residue purified by Sephadex LH20 column
8. Synthesis of telomeric substrates 11a, 12a, 13a
(2.5ꢄ
the desired fractions (detected on TLC by charring with
a methanolꢀsulfuric acid solution) were evaporated
/
35 cm, eluent: CH2Cl2ꢀ
/
MeOH, 50:50, v/v). Then,
8.1. General procedure
/
under reduce pressure. At this point of the synthesis,
the coupling percentage of the peptidic sequence was
specified by comparing in 1H-NMR peaks area assigned
to the aromatic Mtr proton (6.6 ppm) to the terminal
methyl signal in the hydrocarbon tail of the telomer (0.9
ppm). It is here noteworthy that for each telomer 12 and
13, the NMR structural evaluation showed that the
proportion of RGD moieties was similar to the succi-
nimidyl groups one in the precursor telomers 12a or 13a.
Finally, the hydrolysis of both tert-butyl and Mtr
protective groups, was performed using a trifluoroacetic
Monomers 7, 8, 9 and/or 10 were reacted in anhy-
drous DMF (2 mL) (or THF). The different ratio of
different monomers used for the synthesis of telomers
are indicated with the yield of reactions in Table I.
Different amounts of octanethiol telogen (1/Roꢂ
[Monomer]/[Telogen]) as indicated in Table I in the
presence of AIBN ([AIBN]ꢂ[Telogen]/2) were then
/
/
added and the mixture refluxed for at least 12 h until
the complete disappearance of 7, 8, 9 or 10 (monitored
by TLC and detected to UV light). At about the middle
of the reaction, further addition of AIBN ([Telogen]/2)
was effected. When all the starting monomers were
reacted, the solution was concentrated under vacuum
and the mixture was fractionated with a Sephadex LH20
acidꢀ
/
methylene chlorideꢀmethyl phenyl sulfide mixture
/
(3/5/2 v/v/v) at r.t. for 24 h. The solution was then
evaporated under vacuum and precipitated in diethyl
ether until trifluoroacetic acid was evaporated. The
mixture was fractionated with a Sephadex G50 column
column (2.5ꢄ
/
35 cm, eluent: CH2Cl2ꢀMeOH, 50:50, v/
/
v) and the desired fractions (detected on TLC by
(2.5ꢄ35 cm) and the desired fractions (detected on TLC
/

S. Jasseron et al. / European Journal of Medicinal Chemistry 38 (2003) 825ꢀ
/836
835
by charring with a methanolꢀ/sulfuric acid solution)
10. Biodisposition studies
were lyophilized to give a set of telomers 12 as a white
powder (0.103 g, 70%).
10.1. Radioactive labelling
Molecules were labelled with 125I on the tyrosine
moiety using the chloramine T method [45]. Purification
of the labelled compounds was performed by ion
exchange HPLC on anionic column Poros HQM, using
a Biocad Sprint apparatus (Perseptive Biosystems).
Specific radioactivities were respectively 125 mCi mgꢃ1
for 11 and 12 and 135 mCi mgꢃ1 for 13.
9. Pharmacological assays
9.1. B16 cells culture
The murine B16 melanoma cells were obtained from
ICIG (Villejuif, France). Stock cell cultures were main-
tained as monolayers in 75 cm2 culture flasks in
GlutamaxTM (Eagle’s minimum essential medium,
Gibco, Paisly, Scotland) supplemented with 10% foetal
10.2. Animal experiments
C57Bl6 mice were inoculated with 106 B16 cells by
dorsal subcutaneous injection. Animals were used when
the tumour diameter reached around 0.5 cm, then they
received by intravenous injection 20 mCi of labelled
compound dissolved in 100 mL of NaCl 0.9%. Mice were
sacrificed at 15 min, 1 and 6 h post administration of the
drug and immediately frozen in liquid nitrogen. After-
ward, the whole body was sliced (40 mm) using a
cryomicrotome. Tissue radioactive concentration was
determined with a computer-controlled multi-wire pro-
portional counter (Ambis 4000, B. Braun Scencetec).
The number of particles per time and surface unit
obtained with the gaseous detector is a linear function
of the radioisotope concentration. Counts are recorded
in 1.064.448 discrete detection points from which the
composite image is displayed on a high resolution color
monitor so that regions of interest may be directly
evaluated and radioactive concentration can be ex-
pressed as percentage of the administered dose per g
of tissue [46].
calf serum (Bio West, Nuaille, France), 100ꢄ
solution (Gibco), 100 mM sodium pyruvate (Gibco),
100ꢄ non essential amino acids (Gibco) and gentamy-
/
vitamin
/
cin sulfate (Gibco).
9.2. B16 cells cytotoxicty assay
For the cytotoxicity assay, B16 cells were plated into
60 mm Petri dishes (150 cells/dish) and allowed to
adhere for 20 h before treatment. Then, medium was
removed and replaced by new medium containing
increasing concentrations of 11 and 13 from 0.01 to 1
mM and the cells were grown for 10 days at 37 8C in a
CO2 incubator. After this time, the dishes were rinsed
with phosphate buffered saline 0.05 M pH 7.4, cells were
fixed with methanol and stained with 0.2% crystal violet
solution. Colonies of more than 50 cells were counted.
The surviving fraction was calculated as the ratio of
coloning efficiencies of treated and untreated cells. The
antiproliferative activity of the drugs, expressed as IC50,
corresponds to the drug concentration giving a 50%
coloning efficiency compared with untreated cells
[42,43].
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