NC-100717: A versatile RGD peptide scaffold for angiogenesis imaging

Article abstract of DOI:10.1016/j.bmcl.2006.09.033

Targeting the molecular pathways associated with angiogenesis offers great potential in detecting disease pathology using in vivo imaging technologies. Initiation of angiogenesis requires activation and migration of endothelial cells in order for neovascu

Full text of DOI:10.1016/j.bmcl.2006.09.033

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6190–6193
NC-100717: A versatile RGD peptide scaffold
for angiogenesis imaging
˚
Bard Indrevoll, Grete Mørk Kindberg, Magne Solbakken, Emma Bjurgert,
John Henrik Johansen, Hege Karlsen, Marivi Mendizabal and Alan Cuthbertson*
GE Healthcare, Medical Diagnostics, Discovery Research, Oslo, Norway
Received 6 September 2006; accepted 9 September 2006
Available online 26 September 2006
Abstract—Targeting the molecular pathways associated with angiogenesis offers great potential in detecting disease pathology using
in vivo imaging technologies. Initiation of angiogenesis requires activation and migration of endothelial cells in order for neovas-
cularization to proceed. Endothelial cells associate with the extracellular matrix through specific interactions with a variety of cell
adhesion receptors known as integrins. Peptides containing the tripeptide sequence RGD are known to bind with high affinity to the
avb3 and avb5 integrins associated with angiogenesis. We present herein the synthesis and in vitro binding affinity of the RGD-con-
taining peptide NC-100717 and a range of molecular probes derived from this intermediate.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The targeting of biological pathways using molecular
imaging probes holds great promise as a means of
detecting and diagnosing disease at a much earlier stage
than is the norm today. Angiogenesis, the growth of new
blood vessels, is a common pathology associated with a
wide range of diseases requiring activation and migra-
tion of endothelial cells before neovascularization can
proceed.1 Endothelial cell interactions with the extracel-
lular matrix are facilitated by cell adhesion receptors
known as integrins.2,3 Studies have demonstrated upreg-
ulation of avb3/avb5 integrins in the new blood vessels
associated with several diseases including wound heal-
ing, tumour growth, diabetic retinopathy, and macular
degeneration. Peptide ligands containing the tripeptide
sequence RGD are known to possess high affinity for
these integrins and several reports have been published
relating to their utility in molecular imaging.4
on determining the binding affinities of each isomer in
order to select the optimal pairing to generate further
SAR. In an in vitro assay using membranes derived
from the human endothelial adenocarcinoma cell line
EA-Hy 926 we found that the nested configuration,
comprising the Cys2–10; Cys4–8 pairing, peptide 1, had
highest affinity for avb3/avb5. The observed Ki was cal-
culated to be 1.6 nM in competition with radioactive
[
125I]-Echistatin, an RGD-containing peptide isolated
from snake venom. This configuration was therefore
selected as starting point for more extensive synthetic
work. The large-scale synthesis of peptides with multiple
disulfide bridges however still remains a significant syn-
thetic challenge. In addition, the possibility of disulfide
scrambling led us to select a strategy directed at reduc-
ing the complexity of peptide 1 by replacing the outer
disulfide bond with a chemically stable bridge. We pos-
tulated that replacement of the N-terminal Ala1 and
Cys2 residues with the chloroacetyl moiety followed by
selective cyclization with the thiol group of Cys10 would
yield a stable thioether bond with minimal disruption to
the RGD pharmacophore. This was also considered a
way of stabilizing the N-terminus to aminopeptidase
activity in vivo. Substitution of Asp3 with Lys intro-
duced a side-chain amino group as an option for further
derivitization with imaging reporters. Finally a short
PEG-like spacer at the C-terminus had the dual function
of biomodifier and contributed to further stabilize
against carboxypeptidase attack.
In our search for a robust RGD pharmacophore we
were drawn to the peptide ACDCRGDCFCG derived
from a phage display library.5 This peptide was reported
to be a potent and selective binder to both avb3 and
avb5 integrins. With four cysteine residues three possi-
ble disulfide isomers exist. Our initial efforts focussed
Keywords: Angiogenesis; RGD peptide; Molecular imaging; Integrins;
Targeting.
*
Corresponding author. Tel.: +4723185761; fax: +4723186014; e-mail:
alan.cuthbertson@ge.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.033

B. Indrevoll et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6190–6193
6191
Scheme 1 shows the synthetic route chosen for the syn-
thesis of the key intermediate NC-100717, 3. The
peptide, Lys(Boc)-Cys(t-Bu)-Arg(Pmc)-Gly-Asp(O-t-Bu)-
Cys(t-Bu)-Phe-Cys(Trt), was assembled using Fmoc
chemistry on Rink Amide AM resin (0.25 mmol scale)
preloaded with 17-(Fmoc-amino)-5-oxo-6-aza-3,9,12,
15-tetraoxaheptadecanoic acid using 40 min HBTU/
HOBt couplings on an ABI 433A automated peptide
synthesizer.6 The amino-peptidyl resin was N-chloro-
acetylated by treatment with chloroacetic acid anhy-
dride (1 mmol) in DMF for 30 min followed by
extensive washing with DMF, DCM and diethyl ether.
Partially protected peptide was liberated from the solid
support by acidolysis (2 h) in trifluoroacetic acid
(TFA) containing 5% triisopropylsilane, 5% water and
2.5% phenol. Following removal of excess TFA in vacuo
the crude peptide was triturated with diethyl ether and
dried.
to cystine of two t-butyl-protected cysteine residues in
peptide synthesis.7 Purification of the final product
was carried out by preparative HPLC using a Phenom-
enex Luna C18 250 · 50 mm column and a 0–30%
water/acetonitrile gradient over 60 min at a flow rate
of 50 ml/min. Fractions were analysed by LC–MS and
those with a purity of >95% combined and lyophilized.
150 mg of peptide was obtained, in a yield of 52% from
the crude starting product.
Molecular imaging of protein receptor systems in vivo
requires the administration by intravenous injection of
an imaging agent with properties that render the agent
visible when placed in an imaging system. There are
many types of reporter groups available to do this
including fluorescent dyes, radioactive and paramagnet-
ic metal-chelate systems and short-lived radioisotopes
such as 18F-Fluorine and 11-Carbon. Versatile strate-
gies for the conjugation of ligand to reporter in a way
which does not drastically alter the binding affinity for
the receptor are a prerequisite for successful targeting.
In addition, the high cost of developing new pharmaceu-
tical agents means that it is desirable to identify interme-
diates which can be used to produce a wide variety of
imaging probes. To this end we chose the lysine residue
as attachment point for the reporter group. By reaction
of the e-amino function of NC-100717 with the reporter
using amide bond coupling chemistries conjugates could
be prepared readily in good yield and purity.
In a typical experiment peptide 2 was obtained by dis-
solving 322 mg of the crude partially protected peptide
in 50% acetonitrile/water (1 mg/mL) and adjusting the
pH to 8 by addition of dilute ammonia solution. Thio-
ether bridge formation was complete after 16 h at room
temperature as evidenced by LC–MS. The reaction mix-
ture was then dried by lyophilization and conversion to
peptide 3 carried out directly on the crude product by
addition of TFA containing 5% DMSO (3 mg/mL) with
stirring for 15 min at room temperature. The excess
TFA was removed in vacuo and crude final product pre-
cipitated by addition of cold diethyl ether. We have pre-
viously reported this method as a useful procedure to
facilitate the rapid cleavage with simultaneous oxidation
Figure 1 shows the structures of several conjugates de-
rived from NC-100717. Peptide 4 was formed in good
yield by the reaction of 3 with the commercially available
Scheme 1. Synthesis of NC-100717 (3) using a ‘one pot’ protocol for the formation of the two bridges.

6192
B. Indrevoll et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6190–6193
Figure 1. The structures of four imaging probes derived from the core pharmacophore NC-100717.
active ester of the cyanine dye Cy5.5.8 The dye conjugate
has been used in vivo for the fluorescent detection of
Lewis Lung carcinoma in mice using an optical imaging
camera system. Peptide 5 was prepared in three steps
from 3 first by amide bond coupling of the (Boc-amino-
oxy)-PEG-diglycolic acid unit followed by Boc cleavage
in TFA liberating the free aminooxy group and finally
hydrazone formation at pH 3.5 following addition of
4-fluorobenzaldehyde.9 This chemistry is particularly
useful for labelling peptides with radioactive 18F-Fluo-
rine for Positron Emission Tomography (PET) Imaging
and has shown utility in vivo for angiogenesis imaging.10
Peptide 6 was prepared from 3 by reaction with the tetra-
fluorophenol ester of the diamine dioxime chelate.11 The
peptide chelate conjugate was labelled with 99m-Techne-
tium for use in Single Photon Emission Computed
Tomographic (SPECT) Imaging. The 99mTc complex
has been used successfully for detection of malignant le-
sions in patients with breast cancer.12 Finally, 9.6 mg of
the N-hydroxysuccinimide ester of tetraazacyclodode-
canetetraacetic acid (DOTA) was reacted with 20 mg of
3 in 5 ml DMF for 16 h. Following evaporation of
DMF in vacuo the crude product was re-dissolved in
water and peptide 7 isolated by preparative HPLC.
DOTA is a useful chelate for complexation of gallium-
68 (68 Ga). Studies using eluate from a Germanium/Gal-
lium generator system have demonstrated labelling yields
of >90% and work is on-going to study the utility of this
PET radionuclide in angiogenesis imaging. Peptides 1
and 3–8 were purified by HPLC and analysed by LC–
MS.13 The core peptide 3 and peptide 1 were further ana-
lysed by amino acid analysis.14
Table 1. In vitro assay results for compounds 1, 3–8 in the avb3/avb5
EA-Hy 926 membrane assay
Peptide numbers
avb3/avb5 in vitro assay Ki, nMa
1
3
4
5
6
7
8
1.6 ( 1.6)
6.8 ( 0.2)
0.75 ( 0.06)
8.2 ( 2.4)
3.1 ( 1.6)
3.2 ( 2.5)
NDA
a Values are means of three experiments, standard deviation is given in
parentheses; NDA, no detectable activity.

B. Indrevoll et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6190–6193
6193
9. Cuthbertson, A.; Arbo, B.; Gibson, A.; Solbakken, M.
Patent WO 06/ 030291, 2006.
10. Cuthbertson, A.; Kindberg, G.M.; Indrevoll, B.; Arukwe,
J.; Karlsen, H.; Gibson, A.; Davis, J.; Mendizabal, M.;
Morrison, M.; Glaser, M. In Proceedings of the 19th APS
2005, 665.
Table 1 summarizes the in vitro Ki data calculated from
competition experiments with [125I]-Echistatin on EA-
Hy 926 membrane fractions.15 All peptides tested had
binding affinities in the low nanomolar range except
for the negative control peptide 8, a scrambled isomer
prepared by replacing the RGDCF sequence of 3 with
GDFCR.
11. Cuthbertson, A.; Indrevoll, B.; Solbakken, M. Patent WO
03/006491, 2003.
12. Bach-Gansmo, T.; Danielsson, R.; Saracco, A.; Wilcek,
˚
In summary, we present herein a robust and flexible
RGD-containing peptide NC-100717, with affinity for
the integrin receptors avb3/avb5. We have demonstrated
that modification of this flexible scaffold by amide bond
formation at the e-amino group of lysine has limited im-
pact on affinity. We have also shown that a range of
molecular imaging probes with application spanning sev-
eral imaging modalities are readily accessible using this
peptide. The ultimate goal is to develop imaging probes
with clinical utility for the early detection and diagnosis
of disease where angiogenesis plays a significant role.
B.; Bogsrud, T.; Fangberget, A.; Tangerud, A.; Tobin, T.
J. Nucl. Med. 2006, 47, 1434.
13. Peptides 3–8 were characterised by LC–MS on a Thermo
Finnigan instrument using positive mode ESI and peptide
1 on a Thermo Bioanalysis Lasermat MALDI instrument.
The analysis of peptides 3 and 6 was performed using a
gradient of 0–30% B over 10 min on a Phenomenex Luna
5 lm, C18, 50 · 4.6 mm column at a flow rate of 2 mL/
min; peptides 1, 4 and 7 were analysed using a gradient of
10–50% B over 20 min on a Vydac218TP54, 5 lm,
250 · 4.6 mm column at a flow of 1 mL/min; peptides 5
and 8 were analysed using a gradient of 5–50% B over
10 min on Phenomenex Luna 5 lm, C18, 50 · 2 mm
column at a flow of 0.3 mL/min; where A = 0.1% TFA/
water and B = 0.1% TFA/acetonitrile. Peptide 1: tR:
16.3 min, expected [MH+]: 1148: found [MH+] 1148;
amino acid analysis, Ala 1.0 (1.0) Asp 2.0 (2.0), Arg 1.0
(1.0), half-cystine 3.7 (4.0), Gly 2.0 (2.0), Phe 1.0 (1.0);
peptide 3: tR: 6.9 min, expected [MH+]: 1258.5; found
[MH+]: 1258.8; Amino acid analysis, Asp 1.0 (1.0), Arg 1.0
(1.0), half-cystine 2.1 (2.0), Gly 1.0 (1.0), Lys 1.0 (1.0), Phe
1.0 (1.0); peptide 4: tR: 13.9 min, expected [MH+]: 2156.7;
found [MH+]: 2156.4; peptide 5: tR: 8.4 min, expected
[MH+]: 1815.7; found [MH+]: 1815.8; peptide 6: tR:
7.9 min, expected [MH+]: 1697.8; found [MH+]: 1698.0;
peptide 7: tR: 9.7 min, expected [MH+]: 1644.7; found
[MH+]: 1645.2; peptide 8: tR: 5.3 min, expected [MH+]:
1258.5; found [MH+]: 1258.6.
Acknowledgment
Many thanks to Irina Velikyan at the Uppsala Imanet
PET Centre for her work on 68-Gallium labelling of
the DOTA-RGD peptide.
References and notes
1. Carmeliet, P.; Jain, R. K. Nature 2000, 407, 249.
2. Glukhova, M.; Deugnier, M. A.; Thiery, J. P. Mol. Med.
Today 1995, 1, 84.
3. Brooks, P. C.; Cheresh, D. A. Eur. J. Cancer 1996, 32,
2423.
4. Haubner, R.; Wester, H.-J. Curr. Pharm. Design 2004, 10,
1439.
5. Koivunen, E.; Wang, B.; Ruoslahti, E. BioTechnology
1995, 13, 265.
6. Atherton, E.; Sheppard, R. Solid-Phase Synthesis; IRL
Press: Oxford, 1989.
7. Cuthbertson, A.; Indrevoll, B. Org. Lett. 2003, 5, 2954.
8. Cuthbertson, A.; Nairne, J. Patent WO 05/ 123768, 2005.
14. Amino acid analysis was performed by Aminosyraanaly-
scentralen, University of Uppsala, Sweden.
15. Membrane fractions were prepared from EA-Hy 926 cells
using a Polytron homogenizer and isolation by ultracen-
trifugation. The observed Kd was calculated to be 0.2 nM
for [125I]-Echistatin. Peptides were dissolved in phosphate
buffer and serial dilutions tested for competition against
[
125I]-Echistatin. Kis were calculated from the binding
curve using Prism Software.