Novel DOTA-based prochelator for divalent peptide vectorization: Synthesis of dimeric bombesin analogues for multimodality tumor imaging and therapy

Article abstract of DOI:10.1039/b805281f

Dimeric peptidic vectors, obtained by the divalent grafting of bombesin analogues on a newly synthesized DOTA-based prochelator, showed improved qualities as tumor targeted imaging probes in comparison to their monomeric analogues. The Royal Society of Chemistry.

Full text of DOI:10.1039/b805281f

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Novel DOTA-based prochelator for divalent peptide vectorization:
synthesis of dimeric bombesin analogues for multimodality tumor
imaging and therapyw
Keelara Abiraj,a Hugues Jaccard,b Martin Kretzschmar,a Lothar Helmb
and Helmut R. Maecke*a
Received (in Cambridge, UK) 28th March 2008, Accepted 24th April 2008
First published as an Advance Article on the web 20th June 2008
DOI: 10.1039/b805281f
Dimeric peptidic vectors, obtained by the divalent grafting of
bombesin analogues on a newly synthesized DOTA-based pro-
chelator, showed improved qualities as tumor targeted imaging
probes in comparison to their monomeric analogues.
stable complexes with a large number of metal ions, which are
used in different fields of molecular imaging like Gd3+ for
MRI,10 111In and 67Ga for SPECT,2 68Ga and 64Cu for PET11
and Eu3+and Tb3+ for optical imaging.12 Thus, the synthesis
of DOTA-based prochelators for the multivalent grafting of
targeting ligands is of significant interest to develop improved
targeting vectors that could find application in different ima-
ging modalities. Here we report the synthesis of a new DOTA-
based prochelator and its application for the divalent con-
jugation of bombesin analogues. The divalent conjugates were
labeled with 177Lu (as a Gd3+ surrogate) and in vitro assays
were performed using PC-3 (human prostate adenocarcinoma)
cells to compare the tumor cell uptake and cellular retention
with their corresponding monovalent analogues.
Several malignant human tumors overexpress various peptide
hormone receptors on their cell surface.1 These receptors have
become increasingly important as targets for molecular ima-
ging and targeted radionuclide therapy of tumors using re-
ceptor-avid small peptides.2 The expression of somatostatin
receptors in neuroendocrine tumors has been successfully
exploited for the imaging and therapy of somatostatin recep-
tor expressing tumors using radiolabeled somatostatin analo-
gues in a clinical setting.2,3 Several other peptide receptors
such as cholecystokinin, neurotensin and bombesin receptors
have also attracted considerable interest in recent years.4
Bombesin receptors are of particular interest as they are
overexpressed in several of the more prevalent cancers includ-
ing prostate, breast and small cell lung cancer.5
In addition, relaxivity measurements of the gadolinium
complexes of the monovalent and divalent conjugates were
performed to assess their potency as targeted MRI contrast
agents. The synthesis of prochelator (04) containing two free
carboxylic acid groups for the divalent vectorization of target-
ing peptides started from commercially available DO2A-tert-
butyl ester (01) (Scheme 1). N-alkylation of 01 with 2 equiva-
lents of (R/S)-a-bromoglutaric acid 1-tert-butyl ester 5-benzyl
ester (02)13 yielded 03 in 63%. Subsequent hydrogenation of
03 over 10% Pd–C catalyst followed by chromatographic
purification yielded the prochelator 04 in 86%, which was
further characterized by using ESI-MS and NMR (1D-1H,
A high and specific uptake of imaging probe/targeted drug
in the tumor is crucial for effective imaging/therapy. To
achieve this goal, design and synthesis of peptidyl ligands
having high receptor affinity and selectivity is essential. Ap-
plication of multivalent ligands showed markedly increased
binding affinity with their respective receptors compared to
monovalent ligands.6 The multivalent concept has been used
for the development of targeted radiotracers for tumor ima-
ging.7 Extensive studies on radiolabeled multimeric cyclic
RGD peptides demonstrated that an increase in peptide multi-
plicity can significantly enhance the integrin avb3 binding
affinity and improve the tumor targeting capability of the
radiotracer.8
1
1H-1H-COSY, 1D-13C and H-13C-HSQC experiments). The
new prochelator 04 exists in different isomeric forms (RR, SS,
RS & SR) as it was synthesized by using racemic 02.14 The
prochelator 05 containing one free carboxylic acid group was
synthesized following the procedure described earlier.13
The application of prochelator 04 for the divalent vectoriza-
tion with peptide ligands has been demonstrated by synthesiz-
ing divalent bombesin analogues (Scheme 1). In a typical
reaction, two equivalents of bombesin peptide was added to
prochelator 04, dissolved in DMF and pre-activated with
HATU and the pH was adjusted to 8 using DIPEA. After
overnight coupling at room temperature, the DMF was evapo-
rated and the crude mixture was directly deprotected by adding
TFA/TA/H2O/TIPS (95 : 3 : 1 : 1, v/v/v). The crude divalent
peptide conjugates were purified by HPLC (yield 48 to 55%)
and characterized by ESI-MS and MALDI. Analysis of HPLC
fractions by MALDI-MS revealed the presence of considerable
quantities of undesired modified divalent peptide conjugates
produced by the HATU mediated nitrile formation via dehy-
dration of the carboxamide side chain of asparagine and
Because of its excellent chelating properties and the high
in vivo and in vitro stabilities of the corresponding metal
complexes, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tic acid (DOTA) is widely employed for the conjugation of
reporter molecules with targeting ligands.9 DOTA forms very
a Division of Radiological Chemistry, Department of Radiology,
University Hospital Basel, Petersgraben 4, CH-4031 Basel,
Switzerland. E-mail: hmaecke@uhbs.ch; Fax: +41 61 2655559;
Tel: +41 61 2654699
b Laboratoire de Chimie Inorganique et Bioinorganique, Ecole
´
Polytechnique Federale de Lausanne, EPFL-BCH, CH-1015
Lausanne, Switzerland
´
w Electronic supplementary information (ESI) available: Details of
synthesis and characterization of compounds 03, 04 and 06–09 and
in vitro experiments. See DOI: 10.1039/b805281f
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Scheme 1 Synthesis of DOTA-based prochelators and their conjugation to bombesin analogues. (a) K2CO3, AcCN, 18 h, RT; (b) 10% Pd–C/H2,
MeOH, 24 h, RT; (c) Peptide, HATU, DIPEA, 12 h; (d) TFA/TA/H2O/TIPS (95 : 3 : 1 : 1, v/v/v), 8 h.
glutamine.15 The divalent peptide conjugates showed excellent
labeling properties with many radiometals of interest such as
177Lu, 111In, and 68Ga affording a radiolabeling yield 499.5%
at a specific activity of 450 GBq mmolÀ1. Further, the facile
complexation of these conjugates with GdIII illustrates their
potential application as targeted MRI contrast agents.
conjugates have probably one inner-sphere water molecule
and exhibited significant relaxivities at 60 MHz, ranging from
9.3 to 19.2 mMÀ1 sÀ1 (at 25 1C) and 6.9 to 14.7 mMÀ1 sÀ1 (at
37 1C). The values represent the 2- to 4-fold enhancement of
relaxivity relative to Dotarems (Gd-DOTA, the clinically
used MRI contrast agent with a relaxivity of 4.1 mMÀ1 sÀ1
(at 25 1C) and 3.0 mMÀ1 sÀ1 (at 37 1C) at 60 MHz).17 It is also
worth noting that the divalent conjugates exhibit a 2-fold
enhanced relaxivity compared to their monovalent analogues.
The initial evaluation of the conjugates as tumor targeting
vectors has been conducted in vitro using the gadolinium
surrogate radiometal 177Lu. The specific tumor cell uptake and
cellular retention of the monovalent and divalent bombesin
conjugates were studied in GRP-receptor expressing PC-3 cells.
All the four radiolabeled conjugates showed high and specific
tumor cell uptake in PC-3 cells as shown in Fig. 1a. After 4 h,
177Lu-06, 177Lu-07, 177Lu-08 and 177Lu-09 showed specific inter-
nalization of 26.52 Æ 0.83%, 18.26 Æ 1.06%, 41.9 Æ 2.14% and
35.91 Æ 1.46%, respectively. Blocking studies performed using
large excess of Tyr4-bombesin demonstrate that the internaliza-
tion was receptor mediated. The data revealed that the divalent
presence of the targeting ligand results in approximately a 2-fold
A key objective in MRI contrast agent research is to devise
high relaxivity contrast agents based on gadolinium. To devise
such high relaxivity systems, macromolecules have been ex-
plored as scaffolds for the presentation of GdIII complexes.
Every attempt to enhance the relaxivity by the covalently
linked high molecular weight macromolecular conjugates is
hindered by inefficient motional coupling or slow water ex-
change rate that quenches the theoretical relaxivity gain.10
Multivalent functionalization of cyclen with a glutaric acid
derivative has potential advantages as it: (a) facilitates multi-
ple attachment of medium molecular weight macromolecules
such as targeting peptides and (b) enhances the relaxivity due
to the reduced tumbling rate of the conjugate and fast water
exchange.16 As a proof of the concept, we have determined the
T1 relaxivities, r1, of the synthesized conjugates at proton
Larmor frequencies of 30, 40 and 60 MHz (Table 1). All the
Table 1 T1 relaxivities of the gadolinium complexes of monovalent and divalent peptide conjugates at multiple proton Larmor frequenciesa
r1/mMÀ1 sÀ1
30 MHz
40 MHz
60 MHz
Conjugate
25 1C
37 1C
25 1C
37 1C
25 1C
37 1C
Gd–06
Gd–07
Gd–08
Gd–09
9.5
11.9
16.5
20.2
7.2
8.9
12.5
15.3
9.4
11.8
16.4
20.1
7.0
8.7
12.4
15.2
9.3
11.7
15.8
19.2
6.9
8.6
12.0
14.7
a
Longitudinal 1H relaxation rate times T1 were measured on Bruker Minispecs mq30 (30 MHz, 0.71 T), mq40 (40 MHz, 0.94 T) and mq60
(60 MHz, 1.41 T).
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Fig. 1 Comparative in vitro evaluation of tumor targeting potential of 177Lu labeled conjugates 06, 07, 08 and 09 with PC-3 cells. Values and
standard deviations are the result of two independent experiments with triplicates in each experiment. (a) Rate of internalization; (b) cellular
retention.
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For cellular retention studies, the radiopeptides were al-
lowed to internalize for 2 h; cells were then washed twice with
PBS and the receptor-bound ligands were removed by washing
with glycine buffer, pH 2.8. Cells were then incubated by
adding fresh medium and the radioactivity retained in the cell
after 10, 20, 30, 60, 120, and 240 min was measured. Fig. 1b
illustrates the cellular retention of these compounds over time,
expressed as the percentage of radioactivity left in the cell from
the total amount internalized. Within 4 h, 49% of 177Lu-06
and 56% of 177Lu-07 were externalized from the cell. Com-
paratively, divalent conjugates exhibit better cellular retention
(77% of 177Lu-08 and 72% of 177Lu-09 retained after 4 h).
In conclusion, the work demonstrates the synthesis of a new
DOTA-based prochelator for the facile divalent conjugation
of targeting biomolecules such as peptides. The improved
tumor targeting capabilities and enhanced relaxivities exhib-
ited by divalent bombesin analogues are highly promising for
the development of these divalent conjugates as potential
targeting probes, which could be employed as both targeted
MRI contrast agents and radiopharmaceuticals.
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3250 | Chem. Commun., 2008, 3248–3250