RGD-cyclam conjugate: Synthesis and potential application for positron emission tomography

Article abstract of DOI:10.1016/j.bmcl.2010.07.114

Cyclam and DOTA-containing positron emission tomography radiotracers were prepared by using a modular chemical strategy based on peptide synthesis and chemoselective ligations. These molecules encompass two functional domains, one a tumour 'homing' domain and the other a chelating ligand for copper allowing nuclear imaging of tumours.

Full text of DOI:10.1016/j.bmcl.2010.07.114

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5422–5425
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
RGD–cyclam conjugate: Synthesis and potential application for positron
emission tomography
a
b
b
b
b
Mathieu Galibert , Zhao-Hui Jin , Takako Furukawa , Toshimitsu Fukumura , Tsuneo Saga ,
b
a
a,
*
Yasuhisa Fujibayashi , Pascal Dumy , Didier Boturyn
Département de Chimie Moléculaire, UMR-5250, CNRS—Université Joseph Fourier, Grenoble, France
Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclam and DOTA-containing positron emission tomography radiotracers were prepared by using a mod-
ular chemical strategy based on peptide synthesis and chemoselective ligations. These molecules encom-
pass two functional domains, one a tumour ‘homing’ domain and the other a chelating ligand for copper
allowing nuclear imaging of tumours.
Received 25 June 2010
Revised 23 July 2010
Accepted 25 July 2010
Available online 3 August 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
RGD conjugate
Cyclam
DOTA
64
Cu PET
In recent years, a series of RGD (Arg-Gly-Asp) peptide conju-
gates was developed for the non-invasive imaging of tumours
through the molecular targeting of
integrin.1 Different tech-
niques were used including positron emission tomography
but not monomeric RGD peptide could form clusters of two inte-
1
0
grins. We also hypothesize that the observed multivalent effect
could arise from a statistical rebinding of tetrameric-RGD peptide
due to the high local concentration of RGD elements.
V 3
a b
2
3
(
PET), single photon emission tomography (SPECT), magnetic res-
To further improve the in vivo imaging quality by enhancing the
signal to noise ratio through the use of tetrameric RGD-containing
scaffolds, we herein report the synthesis and applicability for PET
4
5
onance imaging (MRI), and optical imaging. Among these meth-
odologies, the most sensitive molecular imaging techniques are the
radionuclide-based PET and SPECT imaging modalities. In particu-
6
18
imaging. Several reports have focused on the use of F to label
1
1
lar, PET has emerged as non-invasive imaging technique since the
RGD-containing probes. For instance, molecular imaging using
1
8
most successful commercial radiopharmaceutical 2-deoxy-2-[ F]-
fluoroglucose is routinely used for clinical evaluation (oncological
imaging). At the same time, we and others designed and synthe-
sized multivalent RGD (Arg-Gly-Asp) peptide ligands with en-
V 3 V 5
a b and a b
integrins.7 We have
hanced binding affinity for
shown that tetrameric RGD-containing scaffolds exhibit desirable
8
biological properties for tumour imaging and for targeted drug
9
delivery. These compounds contain a cluster of four copies of a
cyclo[-RGDfK-] monomer grafted onto a cyclic decapeptide scaffold
(
Fig. 1). We recently measured the affinity constant (K
containing compounds for purified integrins. K values rose from
.87 nM for tetrameric RGD-containing compound to 41.70 nM
D
) of RGD-
D
3
1
0
for cyclo[-RGDfK-].
microscopy study of
This result agreed well with electronic
V 3
a b /RGD-peptides. Tetrameric RGD peptide
*
Corresponding author. Address: Département de Chimie Moléculaire, UMR-
5
250, CNRS—Université Joseph Fourier, 570 rue de la chimie, BP53, 38041 Grenoble
cedex 9, France. Tel.: +33 4 56 52 08 32; fax: +33 4 56 52 08 05.
E-mail address: didier.boturyn@ujf-grenoble.fr (D. Boturyn).
Figure 1. Structure of radiolabelled clustered RGD-containing compound.
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.114

M. Galibert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5422–5425
5423
Scheme 1. Reagents and conditions: (a) Standard Fmoc SPPS chemistry; (b) PyBOP, DIPEA, DMF, 30 min; (c) 5 (6 equiv), TFA/H
2
0 (1:9), 1 h; (d) 9 or 11 (5 equiv), DIPEA (pH
9
2
.0), DMF, 2 h, then TFA/TIS/H O (2 vol, 94:3:3), 2 h.
[18
expression in man and therefore this radiotracer can be potentially
F]-galacto-RGD compound can efficiently show the level of
(3877.0) was found in excellent agreement with the calculated val-
ues (3876.9).
V
a b
3
1
1a
used for planning and controlling
a
V
b
3
-targeted therapies.
Cop-
In parallel, succinimidyl esters of Cyclam and DOTA were pre-
pared (Scheme 2). Commercially available Cyclam 7 was coupled
with ethyl bromoacetate and then treated under alkaline condi-
tions to give the acid 8. Cyclam 9 was then obtained by using N-
per-based radiopharmaceuticals are also extensively used for PET.
Cu-radionuclides offer a varying range of half-lives and decay
forms that make copper an ideal radioisotope for PET imaging
64
0
and radiotherapy. We decided to use
= 12.7 h), and RGD-containing compounds that exhibit DOTA
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or Cy-
Cu radiotracer (t1/
hydroxysuccinimide and N,N -dicyclohexylcarbodiimide. Starting
2
with acid 10, we prepared the DOTA compound 11 using the same
synthetic strategy. Finally, peptide intermediate 6 was coupled
with succinimidyl esters of Cyclam or DOTA under mild condition
(pH 8.0) to provide, respectively, the novel RGD-containing com-
pounds 1 and 2 in 70% yields after RP-HPLC. The two conjugates
1 and 2 were characterised without ambiguity by ESI-MS as the
deconvoluted masses were in total agreement with the calculated
values.
(
clam (1,4,8,11-tetraazacyclotetradecane) chelate agents were de-
signed (Fig. 1).
Scheme 1 gives an overview of the route to construct RGD con-
jugates 1–2. A modular synthesis was adopted: stable oxime bonds
were utilized to append aldehyde-bearing RGD motifs to the cyclo-
decapeptide scaffold prior to grafting copper chelators. All peptides
were prepared by means of solid-phase peptide syntheses (SPPS)
according to methods already developed by our group.1 Aminooxy
groups were directly incorporated during the SPPS by using the
building block 3. We have previously shown that the 1-ethoxye-
thylidene protecting group is entirely compatible with the SPPS
and prevents the N-overacylation side reaction obtained with Boc
protecting group.1 The following head-to-tail cyclisation was per-
formed in DMF under high dilution with PyBOP reagent as repor-
As shown in Figure 2 (right chromatogram), the labelling using
the DOTA-containing compound 2 was not complete after 1 h of
2
6
4
2
incubation with CuCl at 37 °C: a significant amount of free cop-
per remained in the reaction mixture. In contrast, the labelling via
cyclam (Fig. 2, left chromatogram) gave no free copper under the
same labelling condition. Labelling efficiencies obtained for radio-
tracers 1 and 2 were approximately 100% and 25%, respectively.
Similar results were also revealed from autoradiography of TLC
plate (see Supplementary data). Therefore, the use of cyclam as
2
7
d
ted providing the fully protected cyclodecapeptide 4. By using a
13
64
similar synthesis scheme, RGD compound 5 bearing a glyoxylyl
aldehyde group at the lysine side chain was prepared. To append
RGD targeting elements to the cyclodecapeptide scaffold, we per-
formed full deprotection of 4 under acidic conditions and its
chemoselective ligation with the peptide 5. RP-HPLC purification
afforded the key intermediate 6 in satisfying 70% yield. Compound
chelating agent for Cu is far more advantageous than the use of
DOTA.
For this reason, we next examined if 64Cu radiotracer 1 is applica-
ble for PET. The evaluation was performed on HEK(b
-negative control) tumour-bearing mice. The PET image at
5 h post-injection showed a strong tumour uptake in mouse who
3 1
) or HEK(b )
V 3
(a b
6
4
6
was characterized by ESI-MS and the observed molecular weight
received a single dose of 3.7 MBq of Cu radiotracer 1 (Fig. 3). As

5
424
M. Galibert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5422–5425
2 2 2 3 3
Scheme 2. Reagents and conditions: (a) BrCH CO Et, Na CO , CH CN, 85 °C, 16 h; (b) NaOH, MeOH, 5 h; (c) NHS (1.4 equiv), DCC (1 equiv), AcOH/dioxane (1:1), 5 h.
Figure 2. HPLC traces at 1 h for 64Cu labelling of 1 (left chromatogram) and 2 (right chromatogram).
V 3 3
a b (+) HEK(b
) tumour. Mouse received tail vein injection (iv) of 3.7 MBq of 64Cu
Figure 3. PET and optical image at 5 h of athymic nude mouse bearing subcutaneous
radiotracer 1 (left) and 3 days later, iv injection of 10 nM of 12 (right). K = kidney, B = bladder, T = tumour.
expected, significant retention of radioactivity was found in kidneys
and bladder. This result corroborates previous findings on Cy5-con-
taining fluorescent probes 12 rapidly evacuated with urine.
Urine samples collected from mice at 4 h post-injection were
analyzed and no free copper or Cu–cyclam complex was detected
but a major compound that we assume to be a partially degraded
peptide and a small amount of radiotracer 1. Detailed in vivo meta-
bolic stability studies will be evaluated in further study.
1
4,15

M. Galibert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5422–5425
5425
After 3 days, the same mouse was injected with RGD-containing
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.114.