Synthesis and characterization of αvβ3- targeting peptidomimetic chelate conjugates for PET and SPECT imaging

Article abstract of DOI:10.1016/j.bmcl.2012.07.024

There is growing interest in small peptidomimetic α_vβ ₃ integrin antagonists that are readily synthesized and characterized and can be easily handled using physiological conditions. Peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino- neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-β-alanine (IAC) was successfully conjugated to 1-(1-carboxy-3-carbo-t-butoxypropyl)-4,7-(carbo-tert- butoxymethyl)-1,4,7-triazacyclononane (NODA-GA(tBu)₃) and 1-(1-carboxy-3-carbotertbutoxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA-GA(tBu)₄) and radiolabeled with ¹¹¹In, ⁶⁷Ga and ²⁰³Pb. Results of a radioimmunoassay demonstrated binding to purified α_vβ₃ integrin when 1-4 equiv of integrin were added to the reaction. Based on this promising result, investigations are moving forward to evaluate the NODA-GA-IAC and DOTA-GA-IAC conjugates for targeting tumor associated angiogenesis and α _vβ₃ integrin positive tumors to define their PET and SPECT imaging qualities as well as their potential for delivery of therapeutic radionuclides.

Full text of DOI:10.1016/j.bmcl.2012.07.024

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5517–5522
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and characterization of
avb3-targeting peptidomimetic chelate
conjugates for PET and SPECT imaging
Young-Seung Kim a, Kido Nwe a, Diane E. Milenic a, Martin W. Brechbiel a, Stanley Satz b,
Kwamena E. Baidoo a,
⇑
a Radioimmune & Inorganic Chemistry Section, ROB, NCI, NIH, 10 Center Drive, MSC-1002, Rm B3B69, Bethesda, MD 20892-1002, USA
b Advanced Imaging Projects, Inc., Doral, FL, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
There is growing interest in small peptidomimetic avb3 integrin antagonists that are readily synthesized
Received 17 May 2012
Revised 5 July 2012
Accepted 6 July 2012
Available online 14 July 2012
and characterized and can be easily handled using physiological conditions. Peptidomimetic
4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]
-aminoethylsulfonyl-amino-b-alanine (IAC) was successfully conjugated to 1-(1-carboxy-3-carbo-t-but-
oxypropyl)-4,7-(carbo-tert-butoxymethyl)-1,4,7-triazacyclononane (NODA-GA(tBu)3) and 1-(1-carboxy-
3-carbotertbutoxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA-GA(tBu)4) and radiolabeled with 111In,
Keywords:
Integrin avb3
NODAGA
DOTAGA
67Ga and 203Pb. Results of a radioimmunoassay demonstrated binding to purified
avb3 integrin when 1–
4 equiv of integrin were added to the reaction. Based on this promising result, investigations are moving for-
ward to evaluate the NODA-GA-IAC and DOTA-GA-IAC conjugates for targeting tumor associated angiogen-
esis and
avb3 integrin positive tumors to define their PET and SPECT imaging qualities as well as their
Peptidomimetics
Antagonist
68Ga
potential for delivery of therapeutic radionuclides.
Published by Elsevier Ltd.
203Pb
PET imaging
SPECT imaging
Integrins are a family of transmembrane glycoproteins with
associated and b subunits forming 25 unique heterodimers that
such peptidomimetic
a
vb3 integrin antagonist, 4-[2-(3,4,5,6-tetra-
a
hydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-aminoethylsulfo-
nyl-amino-b-alanine (IA) was synthesized by Hood et al.5
Subsequent, modification of IA to the corresponding carbamate
derivativesbytheDanthigroupresultedin4-[2-(3,4,5,6-tetrahydro-
pyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta
-1-carbamyl)]-aminoethylsulfonyl-amino-b-alanine (IAC), with a
binding affinity 20 times greater than that of IA.6 A SPECT (single
photon emission computed tomography) imaging study with
111In-DOTA-Bz-SCN-IAC was also performed and tumor was clearly
visualized at 4 h p.i.7 111In-DOTA-Bz-SCN-IAC was prepared using
the bifunctional chelate DOTA-Bz-SCN which differs from the
DOTA-GA described in this study.
Clinically, SPECT and PET (positron emission tomography) play
significant roles allowing noninvasive imaging of internal physio-
logical and biochemical function and pathologies in vivo. While
PET is more expensive, it has significant advantages over SPECT
with respect to its ability to better quantify images. Of metallic
radionuclides currently being investigated for PET applications,
gallium-68 (68Ga) has grown in popularity.8,9 The popularity of
68Ga stems from the ease of on site production from a long lived
generator system (68Ge/68Ga) rather than a cyclotron, and automa-
tion for incorporation into radiolabeled compounds.10 The
67.7 min half-life of 68Ga is an appropriate match to the biological
facilitate adhesion and migration of cells on the extracellular matrix
proteins found in intercellular spaces and basement membranes.1
One of these integrins,
avb3 integrin, interacts with vitronectin,
fibronectin, fibrinogen, thrombospondin, collagen, laminin and
von Willebrand factor. This integrin is over-expressed in tumor in-
duced angiogenic vessels and in various human tumors, but is
found at low levels on epithelial and endothelial cells. It is therefore
a widely recognized target for the development of molecular probes
for imaging angiogenesis and cancer therapy. Towards this end, the
tumor imaging capability of several RGD peptides that act as avb3
integrin antagonists has been demonstrated by several research
groups. Additionally, several of these peptides have been shown
to inhibit tumor angiogenesis and interrupt metastasis in many
models.2–4
There is growing interest in peptidomimetic avb3 integrin antag-
onists composed of a stable core scaffold with basic and acidic
groups that mimic the guanidine and carboxylate pharmacophore
of RGD peptides. Peptidomimetics tend to have higher activity, spec-
ificity and longer duration of action compared to the peptides. One
⇑
Corresponding author. Tel.: +1 301 496 6494; fax: +1 301 402 1923.
E-mail address: baidook@mail.nih.gov (K.E. Baidoo).
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.07.024

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Y.-S. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5517–5522
half-lives of peptides. Gallium(III) typically binds with chelates
possessing multiple anionic oxygen donors preferring a coordina-
tion number of six in an octahedral geometry. Fitting these prefer-
ences, the macrocyclic ligand, 1,4,7-triazacyclononane-1,4,7-
triacetic acid (NOTA), is well established as forming very stable
complexes with a wide variety of metals, Ga(III) being one of
them.11 Several NOTA derivatives have been reported (Fig. 1) for
use in the radiolabeling of proteins and peptides. Recently, Knetsch
et al. reported a 68Ga-labeled NODA-GA-conjugated RGD peptide
([68Ga]NODA-GA-RGD) that showed better tumor to blood ratio
in vivo than the corresponding [68Ga]DOTA-RGD derivative.12
While interest in 68Ga for PET imaging is currently significant,
there are Pb(II) isotopes that have also been of interest for biomed-
ical applications. Specifically, 203Pb and 212Pb are radiometals pos-
sessing favorable properties for use in nuclear medicine for
single photon emission computed tomography (SPECT) imaging
and is suitable for pharmacokinetic and pharmacodynamic tracer
studies. In addition, 203Pb can serve as one half of a potential
matched-pair of radioisotopes when combined with 212Pb for ther-
apeutic applications. 212Lead (t1/2 = 10.6 h) has been studied as an
‘in vivo generator’ of 212Bi (t1/2 = 60 min) to overcome the short
half-life of that daughter isotope. The macrocyclic polyaminocarb-
oxylate chelate DOTA (1,4,7,10-tetraazacyclododecane-N,N0,N00,
N000-tetraacetic acid) labeled with 212Pb provides a complex that
is adequately stable in vivo to sequester the radionuclide.17
The Mäcke group in Switzerland have synthesized a DOTA
derivative analogous to NOTA-GA, 1-(1-carboxy-3-carbotertbut-
oxymethyl)-1,4,7,10-tetraazacyclododecane
(DOTA-GA(tBu)4).22
The DOTA-GA(tBu)4 affords four intact carboxylic acid functional
groups with a free carboxylate side chain ready for conjugation
to the N-terminus of peptides which makes it useful for biomedical
applications.
potential diagnostic and therapeutic applications, respectively.13–
203Lead (t1/2 = 51.9 h) emits a
c-ray (279 keV) that is ideal for
21
O
O
O
HO
HO
HO
N
N
OH
N
OH
OH
3
2
1
N
N
N
N
N
N
O
O
O
O
O
OH
OH
O
OH
HN
O
O
N
NH2
NCS
O
O
HO
N
5
N
N
O
NCS
N
COOH
HO
O
COOH
OH
N
OH
O
4
N
N
O
O
O
N
OH
O
6
N
O
N
O
O
O
OH
Figure 1. Structures of NOTA derivatives. 7-(5-Maleimido-1-ethoxycarbonylphenyl)-1,4,7-triazacyclononane-1,4-diylacetic acid (1), 2-(4-aminobutyl)-1,4,7-triazacyclo-
nonane-1,4,7-triyltriacetic acid (2), nNOTA (3), p-SCN-Bn-NOTA (4), NETA (5), and NODA-GA(tBu)3 (6).

Y.-S. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5517–5522
5519
O
HN
O
O
O
O
N
H
N
N
O
N
O
NHS, EDC
CDCl3
O
N
O
N
O
N
O
N
O
+
HN
O
O
O
O
O
O
HO
S
O
N
N
H
N
H
O
NH2
OH
O
O
O
O
2
IAC
DMF
DIEA
O
NH
O
O
N
N
H
N
O
O
O
NH
N
O
N
3
TFA
O
O
O
O
O
S
OH
O
O
N
H
O
N
N
NH
H
H
O
HO
O
N
N
H
N
OH
O
N
N
O
1
NH
OH
O
O
O
O
S
OH
O
N
N
H
N
H
H
O
Scheme 1. Synthesis of compound 1.
O
O
O
O
O
O
O
O
O
N
N
N
N
EDC, NHS, 87%
CH2Cl2
O
N
O
O
N
O
N
O
N
O
N
OH
O
O
O
O
O
O
5
HN
N
HN
O
HO
OH
OH
IAC, DIEA, 82%
DMF
O
O
N
O
4
N
NH
O
O
O
O
S
OH
N
N
O
O
N
N
N
HN
N
H
H
H
O
O
HN
HO
O
O
O
O
O
O
TFA, HCl (g), 60%
O
N
N
6
NH
O
O
O
S
OH
N
O
N
N
O
N
N
H
H
H
O
O
O
O
Scheme 2. Synthesis of compound 4.

5520
Y.-S. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5517–5522
Figure 2. Radio-HPLC profiles of 111In-1, A; 67Ga-1, B; and 203Pb-4, C.
Figure 3. HPLC profiles of Ga(III)-1 (top) and 67Ga-1 (bottom).
In the present study, the objective was to move beyond the
EDC were dissolved in dichloromethane and the reaction mixture
was stirred for 24 h. The mixture was extracted with saturated
NaCl solution, 5% NaHCO3, and saturated NaCl again. The organic
layer was dried over MgSO4, filtered, and dried under vacuum
resulting in the formation of yellowish oils 2 or 5. The IAC and
2 or 5 were combined in anhydrous DMF and diisopropylethyl-
amine was added to the mixture which was then stirred over-
night at room temperature. Reverse-phase HPLC purification
followed by TFA deprotection yielded 1 or 4, respectively.23,24
To evaluate the radiolabeling efficiency of the NODA-GA and
use of RGD peptides as delivery vectors to the various integrin
targets and explore the utility of IAC for such applications. 68Gal-
lium labeling was investigated for PET applications using NODA-
GA and 67Ga as a surrogate for 68Ga, and 203Pb for SPECT imaging
using DOTA-GA. To this end, IAC was successfully conjugated
to NODA-GA (Scheme 1) and DOTA-GA (Scheme 2) and the con-
jugates were radiolabeled with 111In or 67Ga for the NODA-GA
conjugate and 203Pb for the DOTA-GA conjugate. In brief,
NODA-GA(tBu)3 or DOTA-GA(tBu)4, N-hydroxysuccinimide, and

Y.-S. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5517–5522
5521
Figure 4. HPLC profiles of Pb(II)-4 (top) and 203Pb-4 (bottom).
Table 1
In conclusion, the peptidomimetic
a
vb3 integrin antagonist
Binding of 111In-1, 67Ga-1 and 203Pb-4 to purified
avb3 integrin
(IAC) was conjugated to NODA-GA and DOTA-GA and successfully
radiolabeled with 111In, 67Ga and 203Pb. This promising preliminary
data is fueling further investigation of NODA-GA-IAC and DOTA-
GA-IAC conjugates for targeting tumor associated angiogenesis
Bound (%)
No integrin (111In-1 only)
5.3
66.0
88.1
5.4
0.5
l
M Integrin + 111In-1 (0.47
l
M)
M)
M Integrin + 111In-1 + IAC (20
1
1
l
l
M Integrin + 111In-1 (0.47
l
and
avb3 integrin positive tumors using PET and SPECT imaging.
l
M)
Other potential applications include the use of radionuclides such
No integrin (67Ga-1 only)
2.1
10.7
25.5
43.6
1.6
as 90Y, 177Lu and 212Pb for radiotherapy.
0.5 l l
M Integrin + 67Ga-1 (0.45
M)
1
2
2
l
l
l
M Integrin + 67Ga-1 (0.45
M Integrin + 67Ga-1 (0.45
lM)
Acknowledgement
lM)
M Integrin + 67Ga-1 + IAC (20
l
M)
No integrin (203Pb-4 only)
0.3
10.9
20.0
33.4
0.3
This research was supported by the Intramural Research Pro-
gram of the NIH, National Cancer Institute, Center for Cancer
Research.
0.5 l l
M Integrin + 203Pb-4 (0.5
M)
1
2
2
l
l
l
M Integrin + 203Pb-4 (0.5
M Integrin + 203Pb-4 (0.5
lM)
lM)
M Integrin + 203Pb-4 + IAC (20
l
M)
Supplementary data
DOTA-GA conjugates, 111In, 67Ga and 203Pb were employed to
demonstrate facile formation of complexes with these radionuc-
lides. The NODA-GA conjugate 1 was efficiently radiolabeled
(>90%) with 111In and 67Ga within 30 min (Fig. 2A and B, respec-
tively). The radiolabeling of the DOTA-GA conjugate 4 with 203Pb
was equally efficient (Fig. 2C). Non-radioactive Ga(III)-1 and
Pb(II)-4 were also synthesized in order to characterize the radio-
labeled 67Ga and 203Pb complexes.25,26 Figs. 3 and 4 demonstrate
HPLC profiles of the mixture containing both Ga(III)-1 and 67Ga-1;
and Pb(II)-4 and 203Pb-4, respectively.
Supplementary data (materials and methods, general synthesis
and radiolabeling conditions) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2012.07.024.
References and notes
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A radioimmunoassay was performed to assess the binding abil-
ity of the radiolabeled NODA-GA and DOTA-GA conjugates with
a
vb3 integrin. The 111In-labeled 1 (2 ꢀ 106 cpm, 0.47
l
M), 67Ga-la-
beled 1 (5 ꢀ 105 cpm, 0.45
l
M) or 203Pb-labeled 4 (3 ꢀ 105 cpm,
0.5
man
for 3 h at 37 °C. For non-specific binding, excess IAC (20
lM) was incubated with 0, 0.5, 1.0 and 2.0 lM of purified hu-
a
vb3 integrin (MW 237,000) in a total volume of 25
lL PBS
lM) was
added to the reaction mixture to block binding. The reaction mix-
ture was then separated on a 10 mL Sephadex G50 column using
PBS as eluent. Fractions (0.5 mL) were collected and subsequently
counted in a c-counter. As indicated in Table 1, the labeled conju-
gates bound the integrin to varying degrees. The binding of 111In-1
was greatest followed by 67Ga-1 and then 203Pb-4. In addition,
binding was blocked ꢁ95% by the addition of a 10 to 20-fold molar
excess of the cold IAC to the reaction solution indicating specific
binding of the labeled conjugates. Furthermore, it is worth noting
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that the reactivity of the 111In-1 with
than that reported for 111In-DOTA-IAC (72%).7
avb3 integrin (88%) is higher

5522
Y.-S. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5517–5522
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23. 1H NMR (D2O) d 0.90 (s, 6H), 1.82 (br, 2H), 2.00 (m, 2H), 2.39 (br t, 2H), 2.9–3.4
(m, 20H), 3.49 (m, 6H), 3.53 (m, 2H), 3.80 (m, 3H), 3.88 (br s, 2H), 4.15 (br, 2H),
4.34 (br t, J = 6.0 Hz, 1H), 6.95 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H). 13C
NMR (D2O) d 14.2, 19.8, 35.1, 39.0, 40.4, 51.7, 66.2, 67.1, 114.5, 121.2, 129.5,
153.8. ESI-MS: m/z = 943.3 for [M+H]+, 472.2 for [M+2H]2+ (943.03 Calcd for
18. Chappell, L. L.; Dadachova, E.; Milenic, D. E.; Garmestani, K.; Wu, C.; Brechbiel,
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C39H62N10O15S). Anal. Calcd for C39H62N10O15S (TFA)2(H2O): C 43.43; H 5.59; N
11.77; S 2.61. Found: C 43.56; H 5.69; N 11.08; S 2.61.
24. ESI-MS: m/z = 1042.3 for [MꢂH]ꢂ (1043.46 Calcd for C43H69O17S).
25. ESI-MS: m/z = 1009.2 for [M+H]+, 506.0 for [M+2H]2+ (1008.31 Calcd for
20. Chong, H-S.; Milenic, D. E.; Garmestani, K.; Brady, E. D.; Arora, H.; Pfiester, C.;
Brechbiel, M. W. Nucl. Med. Biol. 2006, 459.
C
39H59N10O15SGa).
26. ESI-MS: m/z = 1248.3 for [M+H]+ (1247.50 Calcd for C43H65N11O17SPb).