Synthesis and preliminary biological assessments of RGD bearing biocompatible telomers

Article abstract of DOI:10.1016/S0960-894X(02)00079-3

Work reported herein deals with the synthesis and preliminary biological assessments of a new class of biocompatible telomeric carriers bearing peptidic RGDSK sequences as tumor cell targeting and tyrosine moieties labelled with ¹²⁵I as in vivo probe. The radioactivity levels obtained in several tissues, after the intravenous injections of these telomers in mice bearing grafted B16 syngenic melanoma showed that the addition of a RGD residue to a telomeric structure confers it an increased affinity for the highly vascularized zone surrounding the tumor.

Full text of DOI:10.1016/S0960-894X(02)00079-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1067–1070
Synthesis and Preliminary Biological Assessments of
RGD Bearing Biocompatible Telomers
a
a
b
Sylvain Jasseron, Christiane Contino-Pe
´
b
pin, Jean Claude Maurizis,
a,
Maryse Rapp and Bernard Pucci *
aLaboratoire de Chimie Bioorganique et des Syst e` mes Mol e´ culaires Vectoriels, Faculte´ des Sciences,
3
3, rue Louis Pasteur, 84000 Avignon, France
INSERM U484, rue Montalembert, BP 184, 63005 Clermont-Ferrand Cedex, France
b
Received 29 October 2001; revised 2 January 2002; accepted 28 January 2002
Abstract—Work reported herein deals with the synthesis and preliminary biological assessments of a new class of biocompatible
telomeric carriers bearing peptidic RGDSK sequences as tumor cell targeting and tyrosine moieties labelled with 125I as in vivo
probe. The radioactivity levels obtained in several tissues, after the intravenous injections of these telomers in mice bearing grafted
B16 syngenic melanoma showed that the addition of a RGD residue to a telomeric structure confers it an increased affinity for the
highly vascularized zone surrounding the tumor. # 2002 Elsevier Science Ltd. All rights reserved.
The preparation of drug bearing macromolecular con-
jugates in which therapeutic molecules are linked to a
polymeric backbone by means of biodegradable cova-
lent bonds has been widely investigated in recent
ability and a better therapeutic index than the parent
7
,8
drugs. Now, it seems necessary to ensure a specific
tumor cell targeting to these prodrugs in order to reduce
the toxicity of the active principle on normal cells. Work
reported herein deals with the synthesis and preliminary
biological assessments of telomeric delivery systems
allowing the selective targeting of proliferative angio-
genic vascular cells that support tumors growth.
1À4
years.
In this field, we have developed researches on
the biomedical potentialities of a new class of low mac-
5
romolecular delivery systems called ‘telomers’. These
compounds are obtained by free radical polymerization
of an acryloyl monomer derived from tris(hydroxy-
methyl)acrylamidomethane THAM 1 in the presence of
an alkane or a perfluoroalkanethiol as a transfer
reagent. Most of the physico-chemical parameters of
these telomers (molecular weight, hydrophilic lipophilic
balance, electric charge) can be adjusted through both
the starting material and the experimental conditions.
Preliminary biological studies have shown that these
multifunctional molecules exhibit good biocompatibility
Among the numerous surface receptors involved in a
pathologic process like tumor metastasis, integrins, a
class of heterodimeric glycoproteins, provided potential
targets for directing chemotherapeutic agents to
9
tumorigenic lesions. In this field, the avb integrin was
3
reported to play a crucial role in the angiogenesis which
is important for the development of metastatic colo-
9
nies. Many integrins can be inhibited by antibodies or
6
making them suitable for drug delivery systems. It is
noteworthy that the biodistribution in rat and mouse of
THAM derived telomers, specified by whole body
small peptides that possess the RGD (Arg-Gly-Asp)
sequence as a common receptor recognition motif.
Grafting this sequence onto the backbone of macro-
molecular prodrugs could result in highly potent and
selective inhibitors of integrins.
6
autoradiography, is quite ubiquitous in all tissues. The
anchorage of various antitumor 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 bioavail-
RGD peptides and small RGD peptidomimetics have
1
0,11
received considerable attention.
Studies of a variety
of linear and cyclic RGD peptides showed that addi-
tional amino acids modulate the activity of the RGD-
containing sequence toward the avb3 receptor. For
instance, hydrophobic amino acids following the aspartic
*
4
Corresponding author. Tel.: +33-4-9014-4442; fax: +33-4-9014-
449; e-mail: bernard.pucci@univ-avignon.fr
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00079-3

1068
S. Jasseron et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1067–1070
acid moiety (i.e., in position 4) increase it.12 However,
the hydrophilic amino acid serine also increases the
chosen to protect the different amino-acids: t-butyl
group for serine and aspartic acid, Mtr group (2,3,6-
trimethyl-4-methoxy-benzenesulfonyl moiety) in the
case of arginine. Compounds 4 and 5 were obtained
with the aid of BOP and TBTU coupling reagents
respectively using TEA and DIEA as bases in methylene
chloride. Pentafluorophenol (PFP) and DCC were both
used to carry out the synthesis of synthons 6 and 8.
Following this pathway, all compounds gave satisfac-
tory elemental analysis and were fully characterized by
NMR spectroscopy and FAB-mass spectrometry. The
RGDSK protected sequence was prepared with an
overall yield of 43%.
1
3
inhibitory activity for the avb receptor. By contrast
3
neither hydrophilic nor hydrophobic substitutions in
1
4
position 5 seem to influence this activity.
Considering all these data and to provide a good integ-
rins accessibility for the RGD moieties, we chose to
graft a peptidic chain containing the RGDSK sequence
to the telomer backbone through a g-aminobutanoic
acid spacer. Furthermore, in order to allow the in vivo
scintigraphic follow-up of the macromolecule, a tyr-
osine polymerizable monomer was used during the pre-
paration of THAM derived cotelomers.
Telomerization experiments (Fig. 2) were carried out by
refluxing THF under a nitrogen atmosphere in the
presence of octanethiol as transfer reagent and AIBN as
radical initiator. The AIBN concentration was roughly
10 times lower than the telogen’s. The number average
degree of polymerization (DPn), equal to the amount of
repeating units (x+y+z), depends on the (telogen)/
(monomers) ratio adjusted through both starting mate-
rials and experimental conditions. The proportions of
monomers 1, 2 and 3 and octanethiol used are reported
in Table 1. Each experiment was continued until the
complete disappearance of the monomers. Telomers
were purified by chromatography through a Sephadex
LH20 column.
Synthesis
Peracetylated THAM monomer 1 was easily synthesized
from tris(hydroxymethyl)acrylamidomethane by using
the conventional reactants acetic anhydride in pyridine
at room temperature for 12 h. Compound 1 was
obtained after recrystallization in an ethyl acetate/hex-
1
5
ꢀ
ane mixture as a white powder (87% yield, F=97.5 C).
Concerning the preparation of the tyrosine derived
7
monomer, N-g-acrylamidobutyric acid was first acti-
vated as its hydroxysuccinimidyl ester 2. Consecutive
coupling of this active ester to O-(t-butyl) t-butyl l-tyro-
sinate was performed in methylene chloride at room
The structures of these cotelomers, that is the relative
proportions of each tyrosine (x), activated butanoic acid
(y) and THAM (z) moiety in the cotelomer and the
ꢀ
temperature and provided monomer 3 (F=116 C, 87%
yield). These three monomers gave satisfactory ele-
mental analysis and were fully characterized by NMR
spectroscopy.
1
DPn of the macromolecule, were determined in
NMR.
H
RGDSK 4 was prepared following conventional meth-
ods of liquid-phase synthesis applying fmoc-strategy
(Fig. 1). In order to perform the final peptide deprotec-
tion by using acid hydrolysis, suitable labile groups were
The relative proportions of each tyrosine (x), active ester
(y) and peracetylated THAM (z) moiety in the cotelomer
11 were specified by comparing the peaks area assigned to
the terminal methyl signal in the hydrocarbon tail (d
0
2
.9 ppm, 3H) to aromatic tyrosine protons (d 6.79 ppm,
xH), to succinimidyl protons (d 2.61 ppm, 4yH) and to
methylene protons of peracetytaled THAM (d 4.30 ppm,
zH).
6
The benzyloxycarbonyl protection of the side-chain
amino group of lysine in RGDSK sequence 4 was
removed by catalytic hydrogenation on Pd/C. Con-
secutive coupling of this peptide 10 to the active ester
residues of the telomer 11 was carried out in methylene
chloride at room temperature for 24 h under nitrogen
atmosphere. The proportion of RGD sequences grafted
1
onto the telomer was specified by comparing in
H
NMR peaks area assigned to the aromatic Mtr proton
d 6.56 ppm, 1yH) to the terminal methyl signal in the
hydrocarbon tail. It is noteworthy that for each telomer
Tel H and Tel Ac), the NMR structural evaluation
(
(
showed that the proportion of RGD moieties was simi-
lar to the succinimidyl groups one in the precursor tel-
omer 11. The hydrolysis of both t-butyl and Mtr
protective groups by using a trifluoroacetic acid/meth-
ylene chloride mixture (3/7 v/v) at room temperature for
1
24 h was checked by H NMR spectrum after a purifica-
tion step and led to the compound Tel Ac. A consecutive
Figure 1. Synthesis of RGDSK sequence.

S. Jasseron et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1067–1070
1069
Figure 2. Synthesis of cotelomers Tel H and Tel Ac.
deacetylation of THAM residues by transesterification
in methanol with a catalytic amount of sodium methyl-
ate was performed to obtain compound Tel H. Finally,
Tel H and Tel Ac were submitted to a chromatography
on Sephadex G25 column, then lyophilized.
Table 1. Physico-chemical data of cotelomers, H and Ac
Initial conditions
monomer]/[telogen]
Telomer
structures
[
Compd
3
2
1
x (Tyr) y (OSu) z (THAM) DPn
125I-labelling of molecules
Tel H
Tel Ac
8
8
3.2
6
8
14
4
5
5
11
9
15
18
31
Each compound was dissolved in water at a concentra-
tion of 2 mg/mL. To 1 mL of solution was added 1 mCi
The concentrations of each monomer 1, 2 or 3 used are expressed in
relation to the octanethiol concentration.
1
25
of
INa (IMS30 Amersham) then 100 mL of Chlor-
amine T (10 mg/mL). After 5 min at room temperature,
the reaction was stopped by addition of 100 mL of
Na SO (10 mg/mL). Labelled compound was purified
by separation of non-reacted iodide by HPLC on anio-
nic column (Poros HQ-M) using a NaCl gradient from
animals were sacrificed by ether inhalation. Tissue
radioactive concentration (Table 2) was determined
with a computer-controlled multi-wire proportional
counter (Ambis 4000) allowing direct quantitation of
2
3
0
to 1 M. Labelling yield was around 60%.
1
6
regions of interest.
Animal experiments
C57Bl6 male mice, weighing 20–25 g and bearing grafted
B16 syngenic melanoma tumors were given intravenously
Results and Discussion
20 mCi of each labelled compound dissolved in 100 mL
NaCl 0.9%. 15 min, 1 h, and 6 h after administration,
Table 2 shows radioactivity levels in several tissues as a
function of time following administration. Both molecules

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S. Jasseron et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1067–1070
Table 2. Distribution of radioactivity in tissues after iv administration of 20mCi of labelled Tel H and Tel Ac
Time after administration
Compd
15 min
1 h
6 h
Tel H
Tel Ac
Tel H
Tel Ac
Tel H
Tel Ac
Tumor
Stroma
Blood
Kidney
Liver
Thyroid
2.04Æ0.74
7.91Æ0.91
5.62Æ1.01
21.2Æ4.51
2.36Æ0.58
18.5Æ1.87
1.74Æ0.65
6.34Æ0.65
3.29Æ0.28
31.4Æ5.26
2.11Æ0.35
8.57Æ1.15
0.81Æ0.18
2.24Æ0.46
1.80Æ0.12
19.5Æ3.21
1.01Æ0.32
78.5Æ12.5
2.14Æ0.59
4.56Æ0.90
1.99Æ0.22
18.5Æ3.19
0.94Æ0.17
54.5Æ10.2
0.30Æ0.09
0.58Æ0.10
0.57Æ0.09
10.8Æ2.24
0.34Æ0.12
97.1Æ17.3
1.44Æ0.27
1.76Æ0.24
1.09Æ0.08
8.01Æ1.54
0.52Æ0.12
75.4Æ15.2
Results, given in percentage of administered dose per g of tissue, are the mean of five experiments ÆSD.
References and Notes
exhibit 15 min after administration high concentration
in kidney, indicating a fast urinary elimination.
1
2
3
1
4
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At the tumor level, stroma was strongly labelled after
1
5 min and radioactivity spreads into tumoral tissue
after 1 h. 6 h after administration, an homogenous
radioactivity distribution was observed into the whole
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´ ´
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These results also show that the acetylated molecule
concentrates in the tissues more slowly than its non-
acetylated homologue and that its elimination rate is
slower. Particularly, concentration of the labelled com-
pound in the tumor is significantly higher after 1 h for
the acetylated molecule Tel Ac. The affinity of both
compounds for the tumor stroma is demonstrated by
the ratio tissue radioactivity/blood radioactivity >1 for
all times following administration.
7
8
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Ziegler, S. I.; Goodman, S. L.; Senekowitsch-Schmidtke, R.;
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In conclusion, our results prove that the addition of a
RGD residue to a telomeric structure leads to an
increased affinity for the highly vascularized zone sur-
rounding the tumor. On the other hand, the increase of
lipophilicity involved by the acetylation reduces the
elimination rate from the tumor. These properties can
be used to conceive carriers able to specifically vectorize
cytotoxic principles towards solid tumors. Work in this
field is currently underway.
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Jonczyk, A.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 7461.
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