Synthesis of functionalised HP-DO3A chelating agents for conjugation to biomolecules

Article abstract of DOI:10.1039/b905369g

Two novel bifunctional chelating agents (BFCAs) based on the structure of 10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid (HP-DO3A) have been designed and synthesized. BFCAs are able to strongly coordinate metals (e.g. Gd(iii)) and radiometals (e.g.⁹⁰Y(iii), ¹⁷⁷Lu(iii) and ¹¹¹In(iii)), and find applications in the synthesis of products for the imaging and treatment of several cancer types. Indeed, these two BFCAs have been employed in solid-phase peptide synthesis (SPPS) and coupled to well-known peptides such as bombesin and RGD analogues in order to target tumor cells. We also report here the conjugation of one of them to Lys ⁸-oxytocin and radiolabeling with ¹¹¹In(iii) to obtain a new radiopharmaceutical product with potential applications in the diagnosis of tumors over-expressing oxytocin receptors.

Full text of DOI:10.1039/b905369g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of functionalised HP-DO3A chelating agents for conjugation to
biomolecules†
Alessandro Barge,a Enrico Cappelletti,b Giancarlo Cravotto,a Aurelia Ferrigato,b Luciano Lattuada,*b
Fabio Marinonib and Lorenzo Teic
Received 17th March 2009, Accepted 15th June 2009
First published as an Advance Article on the web 20th July 2009
DOI: 10.1039/b905369g
Two novel bifunctional chelating agents (BFCAs) based on the structure of 10-(2-hydroxypropyl)-
1,4,7-tetraazacyclododecane-1,4,7-triacetic acid (HP-DO3A) have been designed and synthesized.
BFCAs are able to strongly coordinate metals (e.g. Gd(III)) and radiometals (e.g. 90Y(III), 177Lu(III) and
111In(III)), and find applications in the synthesis of products for the imaging and treatment of several
cancer types. Indeed, these two BFCAs have been employed in solid-phase peptide synthesis (SPPS)
and coupled to well-known peptides such as bombesin and RGD analogues in order to target tumor
cells. We also report here the conjugation of one of them to Lys8-oxytocin and radiolabeling with
111In(III) to obtain a new radiopharmaceutical product with potential applications in the diagnosis of
tumors over-expressing oxytocin receptors.
Etotal = 939.1 keV) and 177Lu (T 1 = 6.71 d, Etotal = 146.7 keV),
Introduction
2
which are widely employed in nuclear medicine protocols.5–8
Bifunctional chelating agents (BFCAs) are versatile building
Almost all macrocyclic BFCAs reported in the literature
blocks for the synthesis of compounds that are used in diagnostic
are based on DOTA or DOTA monoamide (DOTAMA)
and therapeutic medical applications.1–4 A BFCA is composed of:
(a) an acyclic or macrocyclic ligand that chelates the desired metal
derivatives bearing a variety of functional groups available for
conjugation, viz. carboxyls,14,15 amines,16 aldehydes or ketones,16,17
isothiocyanates,18–19 maleimides,16,20 alkynes17 and vinylsulfone.21
Surprisingly, no reports have appeared so far concerning
derivatives of the octadentate macrocyclic ligand HP-DO3A [10-
(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
ion and (b) a suitable reactive moiety that can bind covalently
to a specific targeting vector (TV). This kind of coupling is of
broad interest as it finds numerous applications. For example,
by conjugating a metallic radionuclide to a TV, such as a
monoclonal antibody5 or a peptide6–8, radiopharmaceuticals have
been prepared and successfully employed in oncology. Moreover,
the specificity of contrast agents (CA) used for magnetic resonance
imaging (MRI) can be enhanced by conjugating it to a carrier
that targets a specific epitope.9,10 Because trivalent lanthanide ions
display a variety of magnetic, optical and catalytic properties,11
the design and development of synthetic BFCAs for connecting
TVs to lanthanide complexes, as well as any improvements in
related conjugation and chelation procedures, are matters of great
importance to molecular medicine.
R
acid]. Gd-HPDO3A (Prohance,ꢀ Bracco Imaging), a non-specific
CA for MRI, is commercially available and widely employed
in clinical practice.22 A neutral, highly hydrophilic gadolinium
chelate, it is endowed with a very high thermodynamic stability
(log K = 23.8) and kinetic inertness that ensures against any
significant release of Gd3+ ions under in vivo conditions.12,23 Its
relaxivity, r1p (being the relaxation enhancement of solvent water
protons at a 1 mM concentration of the paramagnetic chelate), is
3.7 mM-1 s-1 in water at 40 ◦C, in line with other macrocyclic Gd
complexes.24
The high affinity shown by trivalent metal ions such as Ln(III),
Ga(III) and In(III) towards some polyaminocarboxylic acids,
such as 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic
acid (DOTA) and its derivatives, has been exploited to prepare
very stable complexes for various applications.12,13 For example,
these polyaminocarboxylic ligands are very useful for the com-
From a radiolabeling standpoint, the formation kinetics of
DOTA chelates leave much to be desired, as either lengthy
radiolabeling protocols and/or the use of elevated temperatures
are required to achieve acceptable yields and specific activities.2,4
The use of the HP-DO3A coordinating cage may bring some
improvement in the radiolabeling of bioconjugates.
In an optimal BFCA, a spacer is inserted between the macro-
cyclic coordination cage and the TV to modulate the phar-
macokinetic properties and biodistribution of the conjugate by
changing its overall charge and hydrophilicity.4,25 For this purpose,
hydrocarbon, PEG or polypeptide spacers are usually employed.
For instance, the macrocyclic chelate, often being bulkier than the
vector itself, may strongly affect both the conjugation reaction
and the bioconjugate interaction with the target; it is therefore
advisable to distance it from the vector moiety.26
plexation of b--emitting radionuclides, such as 90Y (T 1 = 2.67 d,
2
aDipartimento di Scienza e Tecnologia del Farmaco, Universita` di Torino,
Via Giuria 9, 10125 Torino, Italy
bBracco Imaging SpA, Bracco Research Centre, Via Ribes 5, 10010
Colleretto Giacosa (TO), Italy. E-mail: Luciano.Lattuada@bracco.com;
Fax: +390125561870; Tel: +390125561810
cDipartimento di Scienze dell’Ambiente e della Vita, Universita` del Piemonte
Orientale, Via T. Michel 11, 15100 Alessandria, Italy
Herein, we report the synthesis and characterization of two
new BFCAs based on the HP-DO3A chelating unit, featuring
† Electronic supplementary information (ESI) available: HPLC and FPLC
methods; 13C, DEPT and 1H NMR spectra. See DOI: 10.1039/b905369g
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Scheme 1 The synthesis of bifunctional chelating agent 5. Reagents and conditions: (i) Na, THF, BrCH2COONa, room temperature for 16 h then reflux
for 3 h; (ii) BnBr, DBU, toluene, room temperature, 2 h; (iii) 3-chloroperbenzoic acid, CHCl3, room temperature, 48 h; (iv) Et3N, CH3CN, 50 ◦C, 32 h;
(v) H2, 10% Pd/C, MeOH, room temperature, 2 h. Yields are indicated in parentheses.
a free carboxyl group for conjugation to the amino groups
of biomolecules. These BFCAs differ in the spacer interposed
Results and discussion
Ligand synthesis
between the coordinating cage and the carboxyl group; the spacer
is a diethylene glycol unit for compound 5 (Scheme 1) and an
aromatic moiety for compound 8 (Scheme 2). BFCAs 5 and 8
have been successfully employed in solid-phase peptide synthesis
(SPPS),27 and conjugated to well-known peptides (e.g. bombesin28
and cyclic RGD29) that specifically target cells over-expressing
their corresponding receptors.6–8 Moreover, we also exemplify here
the conjugation of 8 to Lys8-oxytocin to obtain, after labelling
with a suitable radionuclide, a product with potential applications
in the diagnosis and therapy of tumors over-expressing oxytocin
receptors.
To synthesize ligand 5, containing the hydrophilic diethylene glycol
spacer, we began by preparing sodium 2-allyloxyethanolate by
deprotonation of 2-allyloxyethanol with sodium metal in THF,
followed by a Wilkinson reaction with bromoacetic acid. The
carboxyl group was then protected as a benzyl ester by a reaction
with benzyl bromide in toluene with 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) as the base. At this point, the terminal alkene
was oxidized in good yield to the correspondent epoxide with
3-chloroperbenzoic acid in CHCl3. To open the epoxide, we used
the secondary amino group of DO3A(tBu)3 as the nucleophile
and triethylamine as the base in warm CH3CN. The alkylated
macrocycle was isolated in 59% yield after purification by flash
chromatography. Finally, 5 was obtained by catalytic hydrogenoly-
sis of the benzyl ester under a hydrogen atmosphere at atmospheric
pressure using Pd/C as the heterogeneous catalyst and MeOH as
the solvent. The five-step synthesis had an overall 12% yield.
Bifunctional chelating ligand 8, containing an aromatic spacer,
was synthesized in a three-step sequence starting from epibro-
mohydrin and benzyl 4-hydroxybenzoate. The resulting epoxide
was reacted with DO3A(tBu)3 to afford the protected precursor
of 8 in good yield after purification by flash chromatography. Pd-
catalyzed hydrogenolysis of the benzyl ester at room temperature
and atmospheric pressure gave the final product in an overall 38%
yield.
Conjugation to peptides and labelling
Since most peptides are prepared by SPPS, it is convenient
to perform the conjugation step on the same solid support.
This approach offers the well-known advantages of the SPPS
technique, i.e. the attachment of the BFCA to a precise position
on the peptide, purification of the conjugate by simple filtra-
tion and washing of the solid resin, simultaneous deprotection
Scheme 2 The synthesis of bifunctional chelating agent 8. Reagents and
conditions: (i) K2CO3, DMF, 70 ◦C, 5 h; (ii) DO3A tris-tert-butyl ester,
CH3CN, reflux, 16 h; (iii) H2, 5% Pd/C, MeOH, room temperature, 8 h.
Yields are indicated in parentheses.
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Scheme 3 The structure of oxytocin conjugates 9 and 10.
of the tert-butyl esters of the BFCA, removal of all the protecting
groups on the peptide and cleavage from the resin. Therefore, 5
and 8 were designed in order to be used in SPPS with in situ-
activation of the free carboxyl group with tetramethyluronium-
type coupling agents such as HATU (N,N,N¢,N¢-tetramethyl-O-
(7-azabenzotriazol-1-yl)uronium hexafluorophosphate) or HBTU
(N,N,N¢,N¢-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hex-
afluorophosphate).
Recently, we reported the coupling of 8 to the terminal NH2
group of BBN[7-14]NH2, a truncated sequence of bombesin
(BBN).28 BBN, a 14-amino acid peptide isolated from frog skin,
is an analogue of the human gastrin-releasing peptide (GRP) that
binds to GRP receptors with a high specificity and affinity. BBN
has been much studied because it is known that several types
of human cancer cells have over-expressed or uniquely expressed
GRP receptors.30,31
is stable under Fmoc deprotection conditions but can be easily
cleaved with a hydrazine solution before coupling to 8. Cleav-
age from the resin with TFA also removed all the protecting
groups; then, the intramolecular disulfide bond was formed by
air oxidation in water/DMSO.36 In order to assess the labelling
ability of conjugate 9 with 111In(III), a conjugate containing a
DOTAMA chelating unit10 instead of HP-DO3A was synthesised
in a similar manner. Both conjugates 9 and 10 were then purified
by preparative HPLC.
A labelling test with 111InCl3 was performed on 9 and 10 in order
to compare the chelating units HPDO3A and DOTA monoamide
for their coordinating ability to 111In(III) (Table 1). Interestingly,
labelling was more efficient with 9 (reaching 94%) than 10 (83%).
Since the spacers usually do not affect the metal complexation,
this difference in labelling efficiency could only be ascribed to the
faster In(III) complexation kinetics for HP-DO3A with respect to
DOTAMA ligands. However, the results from fast protein liquid
chromatography (FPLC) were slightly lower than those obtained
by the Sep-Pak method, although the labelling efficiency was still
higher for 9 than 10 (83 vs. 69%, respectively). Indeed, the Sep-
Pak method is a rapid but rough separation method, and it can
Herein, we report the synthesis of 9, a conjugate of 8 and
Lys8-oxytocin, as well as its labelling with 111In(III) (Scheme 3).
Oxytocin,
a cyclic nonapeptide hormone secreted by the
neurohypophysis, acts on target cells by binding to specific
G-protein-coupled receptors.32 Oxytocin receptors are normally
expressed on endometrial cells, while are over-expressed by
several tumor types affecting organs such as breasts, brain and
endometrium.33 Our synthesis of the conjugate was accomplished
by Fmoc-based SPPS,34 incorporating into the peptide sequence
Fmoc-Lys(ivDde)OH. This orthogonally-protected lysine has
the a-nitrogen protected as 9-fluorenylmethoxycarbonyl (Fmoc)
and the e-nitrogen protected with a 1-(4,4-dimethyl-2,6-dioxo-
cyclohexylidene)-3-methylbutyl group (ivDde).35 The ivDde group
Table 1 Labelling percentages obtained after incubation with 111InCl3 in
0.1 M sodium acetate buffer at 90 ◦C for 30 min, followed by purification
by Sep-Pak or FPLC
Product
Sep-Pak
FPLC
HPDO3A-Lys8-oxytocin (9)
DOTAMA-Lys8-oxytocin (10)
94
83
83
69
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only provide a first rapid estimation of the labelling efficiency.
FPLC provides a more accurate value of the amount of labelled
conjugate.
Preparation of 2-[2-(2-propenyloxy)ethoxy]acetic acid (1). 2-
Allyloxyethanol (56.1 g, 0.55 mol) was dissolved in anhydrous
THF (200 mL); then, metallic sodium (11.5 g, 0.50 mol) was added
in small portions. The mixture was stirred at reflux for 3 h until
complete dissolution of the sodium. At the same time, a solution
of bromoacetic acid (69.9 g, 0.50 mol) in water (150 mL) was
cooled in an ice bath and its pH was adjusted to 7 with 2 N
NaOH (250 mL, 0.5 mol). The solution was then lyophilized and
the solid suspended in anhydrous THF (200 mL). The suspension
was added to the sodium alkoxide solution and the mixture stirred
overnight at room temperature and then refluxed for 3 h. The
solvent was removed, the residue dissolved in water (250 mL) and
the solution extracted first with Et2O (4 ¥ 50 mL) then with CH2Cl2
(4 ¥ 50 mL). The aqueous solution was brought to pH 1 by the
addition of 37% HCl, then extracted with CH2Cl2 (4 ¥ 50 mL). The
organic phase was separated, dried (Na2SO4) and evaporated to
give 1 (38.6 g, 48%) as a white solid. TLC (silica gel 60 F254, 90 : 10
CHCl3/MeOH, detection: 1% KMnO4 in 1 M NaOH): Rf = 0.6;
HPLC (system A): Rt = 9.05 min, purity 88%; 1H NMR (CDCl3):
d 9.21 (b, 1H), 5.92 (m, 1H), 5.28 (dd, 3J = 17.2 Hz, 2J = 3.2 Hz,
1H), 5.20 (dd, 3J = 9.9 Hz, 2J = 3.2 Hz, 1H), 4.18 (s, 2H), 4.05 (m,
Since the linker between the chelating unit and the carboxyl
group in 8 is quite hydrophobic and rigid, 5 was designed to
have a more hydrophilic, as well as more flexible, spacer. Because
angiogenesis has a crucial role in tumor growth, metastasis and
inflammatory diseases, the coupling efficiency of 5 to an avb3
integrin-binding “RGD type” peptide has been the object of
a recent patent.29 In fact, avb3 integrin, a specific endothelial
cell surface marker, is up-regulated in angiogenesis, whereas it
is almost absent in quiescent blood vessels. For this reason it has
been targeted with different imaging probes for molecular imaging
applications.37,38
Conclusions
New bifunctional chelators 5 and 8, based on the HP-DO3A
coordinating cage, have been synthesized, and the different
flexibility and hydrophilicity of their spacers was exploited to
tune the characteristics of the final conjugates. Indeed, these two
BFCAs have been successfully employed in SPPS, and coupled
to a bombesin derivative28 and a cyclic RGD peptide.29 Here, in
particular, we have reported the coupling of 8 to an oxytocin
derivative. Labelling with In(III) proved to be more efficient for HP-
DO3A conjugate 9 than the corresponding DOTAMA conjugate
10. Considering that HP-DO3A gives metal complexes with a
high thermodynamic stability (e.g. log KGdHP-DO3A = 23.8 and
log KYHP-DO3A = 22.2),41 bifunctional chelators 5 and 8 will find
applications in the complexation of other metals (e.g. Gd(III)) or
radiometals (e.g. 90Y(III), 177Lu(III)) to achieve new contrast agents
for MRI and nuclear medicine.
3
2H), 3.77 (t, J = 4.1 Hz, 2H) and 3.64 (t, J = 4.1 Hz, 2H); 13C
NMR (CDCl3): d 174.42, 134.45, 118.15, 72.68, 71.55, 69.51 and
68.91. MS (ESI+): calc. for [C7H12O4+H]+ 161.16, found 161.1;
calc. for [C7H12O4+Na]+ 183.06, found 182.9.
2-[2-(2-Propenyloxy)ethoxy]acetic acid benzyl ester (2). A so-
lution of benzyl bromide (6.57 g, 38.0 mmol) in toluene (30 mL)
was added dropwise to a solution of compound 1 (5.13 g, 32.0
mmol) and DBU (4.87 g, 32.0 mmol) in toluene (70 mL). The
mixture was stirred for 2 h, then filtered and evaporated under
vacuum. The residue was dissolved in CHCl3 (50 mL) and the
solution washed with water (3 ¥ 50 mL). The organic layer
was separated, dried (Na2SO4) and evaporated under vacuum.
The residue was purified by flash chromatography (silica gel
column, eluent: 8 : 2 petroleum ether/EtOAc) to yield compound
2 (5.40 g, 68%) as a yellow oil. TLC (silica gel 60 F254, 80 : 20
n-hexane/EtOAc, detection: 1% KMnO4 in 1 M NaOH): Rf =
0.16; HPLC (system B): Rt = 11.33 min, purity 98.7%; 1H NMR
(CDCl3): d 7.37 (s, 5H), 5.92 (m, 1H), 5.28 (dd, 3J = 17.9 Hz, 2J
= 3.3 Hz, 1H), 5.21 (s, 2H), 5.19 (dd, 3J = 10.0 Hz, 2J = 3.3 Hz,
1H), 4.23 (s, 2H), 4.03 (m, 2H), 3.71 (t, 3J = 4.3 Hz, 2H) and 3.65
(t, 3J = 4.3 Hz, 2H). 13C NMR (CDCl3): d 170.74, 135.88, 135.03,
128.96, 117.58, 77.47, 71.34, 69.89, 69.14 and 66.90. MS (ESI+):
calc. for [C14H18O4+H]+ 273.11, found 273.0.
Experimental
Materials and instrumentation
All chemicals were purchased from Sigma-Aldrich Srl (Milan,
Italy) or Lancaster Synthesis GmbH (Mu¨hlheim am Main,
Germany) and were used without purification. Rink amide
NovaGelTM resin and Fmoc amino acids were purchased
R
from Novabiochemꢀ (Merck KGaA, Darmstadt, Germany).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-
dimethylethyl) ester (DOTA tris-tert-butyl ester)14 and 1,4,7,10-
tetraazacyclododecane-1,4,7-triacetic acid tris(1,1-dimethylethyl)
ester (DO3A tris-tert-butyl ester)39 were prepared by fol-
lowing reported procedures. “Reagent B” is a mixture of
TFA/phenol/water/triisopropylsilane = 88 : 5 : 5 : 2 (v/v).40
Sep-Pak C18 cartridges were purchased from Waters (Vimodrone,
Italy). NMR spectra were recorded on a Bruker Avance 400
instrument (operating at 9.4 T). Mass spectra were recorded with a
ThermoFinnigan TSQ700 triple-quadrupole instrument equipped
with an electrospray ionization source. HPLC was performed on
a Merck-Hitachi L6200 and L6000 system equipped with a L4500
UV detector. FPLC was carried out on a Pharmacia Biotech LCC-
501 system equipped with a UV detector (LKB-UV-MII, Phar-
macia Biotech) and a radiodetector (Flow scintillation analyzer,
Radiomatic 150 TR, Packard, Meriden, CT). All chromatographic
methods are listed in the ESI.†
2-[2-(2-Oxiranylmethoxy)ethoxy]acetic acid phenylmethyl ester
(3). A solution of 3-chloroperbenzoic acid (3.96 g, 23.0 mmol)
in CHCl3 (70 mL) was added dropwise to a solution of compound 2
(5.20 g, 21.0 mmol) in CHCl3 (50 mL). The mixture was stirred for
48 h, then washed with 10% aq. Na2SO3 (3 ¥ 150 mL) and water
(3 ¥ 150 mL). The organic layer was separated, dried (Na2SO4)
and evaporated under vacuum. The residue was purified by flash
chromatography (silica gel column, eluent: 1 : 1 CH2Cl2/Et2O) to
yield compound 3 (4.05 g, 72%) as a yellow oil. TLC (silica gel
60 F254, 80 : 20 n-hexane/EtOAc detection: 1% KMnO4 in 1 M
NaOH): Rf = 0.2; HPLC (system B): Rt = 9.69 min, purity 90%;
1H NMR (CDCl3): d 7.34 (s, 5H), 5.17 (s, 2H), 4.19 (s, 2H), 3.73
(m, 5H), 3.50 (m, 1H), 3.13 (m, 1H), 2.75 (m, 1H) and 2.58 (m,
1H). 13C NMR (CDCl3): d 170.65, 135.87, 128.99, 128.81, 128.79,
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72.36,71.33, 71.13, 69.07, 66.87, 51.13 and 44.56. MS (ESI+): calc.
for [C14H18O5+Na]+ 289.10, found 289.0.
123, 115, 69, 67, 50 and 45. MS (ESI+): calc. for [C17H16O4+Na]+
307.09, found 307.0.
4-[2-Hydroxy-3-[4,7,10-tris[2-(1,1-dimethylethoxy)-2-oxoethyl]-
1,4,7,10-tetrazacyclododec-1-yl]propoxy]benzoic acid phenylmethyl
ester (7). A solution of 6 (1.1 g, 3.7 mmol) and DO3A tris-tert-
butyl ester (1.6 g, 3.1 mmol) in CH3CN (25 mL) was stirred for 16 h
at reflux. The solvent was evaporated, the oily residue dissolved
in CHCl3 (25 mL), washed with H2O (3 ¥ 10 mL) and brine (2 ¥
10 mL), dried (Na2SO4) and evaporated under vacuum. The crude
was purified by flash chromatography (silica gel, CHCl3/MeOH
9 : 1) to afford 7 (1.3 g, 56%) as a pale yellow solid. TLC (silica gel
60 F254, CHCl3/MeOH/NH4OH 6 : 4 : 2, detection: UV 254 nm,
1% KMnO4 in 1 M NaOH): Rf = 0.35; HPLC (system C): Rt
10-[2-Hydroxy-3-[2-[2-oxo-2-(phenylmethoxy)ethoxy]ethoxy]pr-
opyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid 1,4,7-
tris(1,1-dimethylethyl) ester (4). A solution of compound 3
(2.44 g, 9.20 mmol) in CH3CN (30 mL) was added dropwise to
a solution of DO3A tris-tert-butyl ester (3.19 g, 6.20 mmol) and
Et3N (1.27 mL, 9.20 mmol) in CH3CN (30 mL). The mixture was
stirred at 50 ◦C for 32 h, then cooled down to room temperature
and evaporated under vacuum. The residue was dissolved in
CHCl3 (50 mL), and the solution washed with water (50 mL) and
brine (50 mL). The organic layer was separated, dried (Na2SO4)
and evaporated under vacuum. The residue was purified by
flash chromatography (silica gel column, eluent: 90 : 10 : 1
CHCl3/MeOH/NH4OH) to yield compound 4 (2.87 g, 59%) as a
yellow oil. TLC (silica gel 60 F254, 90 : 10 : 1 CHCl3/MeOH/NH3,
detection: 1% KMnO4 in 1 M NaOH): Rf = 0.33; HPLC (system
B): Rt = 9.59 min, purity 81%; 1H NMR (CDCl3): d 7.34 (s, 5H),
5.16 (s, 2H), 4.13 (s, 2H), 3.68 (m, 9H), 3.53 (m, 4H), 3.34 (m,
H) and 3.23 (d, J = 17.1 Hz, 1H). 13C NMR (CDCl3): d 173.20,
172.77, 172.52, 170.54, 129.01,127.41, 82.71, 82.42, 74.52, 71.22,
70.94, 68.98, 66.91, 56.87, 56.36, 55.91, 52.90, 52.60, 50.98, 49.67,
48.91, 28.53 and 28.30. MS (ESI+): calc. for [C40H68N4O11+Na]+
803.48, found 803.5.
1
= 13.32 min, purity 98.3%; H NMR (CDCl3): d 7.96 (d, J =
8.52 Hz, 2H), 7.35 (m, 5H), 6.92 (d, J = 8.52 Hz, 2H), 5.30 (s, 2H),
5.15 (d, J = 7.57 Hz, 1H), 4.35 (m, 1H), 4.30 (br, 1H), 4.01 (m,
1H), 3.75 (d, J = 16.9 Hz, 1H), 3.34 (d, J = 17.9 Hz, 1H), 3.22
(d, J = 17.8 Hz, 1H), 3.10–2.80 (enveloped signals, 10H), 2.52 (t,
J = 12.4 Hz, 1H), 2.41 (t, J = 12.6 Hz, 1H), 2.36–2.21 (enveloped
signals, 4H), 2.20–2.01 (enveloped signals, 5H), 1.46 (s, 18H) and
1.42 (s, 9H). 13C NMR (CDCl3): d 173, 172, 156, 153, 137, 132,
129, 128, 123, 83, 82, 72, 67, 65, 57, 56, 55, 52, 50, 49 and 48. MS
(ESI+): calc. for [C43H66N4O10+H]+ 799.48, found 799.6; calc. for
[C43H66N4NaO10+Na]+ 821.47, found 821.5.
4-[2-Hydroxy-3-[4,7,10-tris[2-(1,1-dimethylethoxy)-2-oxoethyl]-
1,4,7,10-tetrazacyclododec-1-yl]propoxy] benzoic acid (8). 5%
Pd/C (310 mg) was added to a solution of 7 (2.2 g, 2.8 mmol) in
MeOH (50 mL) and the mixture was stirred under a hydrogen
atmosphere at room temperature for 8 h. Then, more 5% Pd/C
(310 mg) was added and the mixture was stirred for a further 8 h.
The catalyst was then filtered off (paper filter, then Millipore FH
0.5 mm filters) and the solvent evaporated to afford 8 (1.7 g, 86%)
as a white solid. TLC (silica gel 60 F254, CHCl3/MeOH/NH4OH
9 : 1 : 0.1, detection: 1% KMnO4 in 1 M NaOH): Rf = 0.15; HPLC
10-[3-[2-(Carboxymethoxy)ethoxy]-2-hydroxypropyl]-1,4,7,10-
tetraazacyclododecane-1,4,7-triacetic acid 1,4,7-tris(1,1-dime-
thylethyl) ester (5). 10% Pd/C (386 mg) was added to a solution
of compound 4 (1.93 g, 2.47 mmol) in MeOH (50 mL). The
reaction mixture was stirred under a hydrogen atmosphere for
R
2 h, then filtered through a Milliporeꢀ apparatus (FH 0.5 mm)
and evaporated to yield 5 (1.53 g; 89%) as a colorless oil.
TLC (silica gel 60 F254, 80 : 20 CHCl3/MeOH, detection: 1%
KMnO4 in 1 M NaOH): Rf = 0.42; HPLC (system B): Rt =
1
3.52 min, purity 90%; H NMR (CDCl3): d 4.34 (br, 2H), 4.13
1
(system C): Rt = 10.12 min, purity 99%; H NMR (CDCl3): d
(m, 3H), 3.76 (m, 2H), 3.69 (br, 10H), 3.51 (br, 2H), 3.47 (s, 4H),
3.43 (br, 2H), 3.37 (br, 2H), 3.09 (br, 4H), 2.86 (br, 4H), 1.47 (s,
9H) and 1.45 (s, 18H). 13C NMR (CDCl3): d 172.77, 170.93, 82.49,
73.33, 71.51, 70.84, 69.52, 64.75, 56.56, 55.27, 53.00, 51.00, 28.53
and 28.49. MS (ESI+): calc. for [C33H62N4O11+H]+ 691.45, found
691.5; calc. for [C33H62N4O11+Na]+ 713.43, found 713.5.
7.76 (br, 2H), 6.70 (br, 2H), 4.67 (br, 2H), 4.42–4.16 (enveloped
signals, 2H), 4.10 (br, 2H), 4.03–3.49 (enveloped signals, 8H), 3.36
(br, 3H), 3.10 (br, 4H), 2.78 (br, 6H) and 1.40 (s, 27H). 13C NMR
(CDCl3): d 171, 162, 132, 123, 114, 82, 70, 58, 57, 55, 53, 52, 50,
48 and 29. MS (ESI+): calc. for [C36H60N4O10+H]+ 709.44, found
709.5; calc. for [C36H60N4O10+Na]+ 731.42, found 731.6.
4-(Oxyranylmethoxy)benzoic acid phenylmethyl ester (6). An-
hydrous K2CO3 (11.8 g, 85.4 mmol) was added to a solution of
benzyl 4-hydroxybenzoate (12.9 g, 56.7 mmol) and 1-bromo-2,3-
epoxypropane (10.5 g, 76.7 mmol) in N,N-dimethylformamide
(DMF) (60 mL). The mixture was stirred for 5 h at 70 ◦C and 12 h at
room temperature. The solvent was then evaporated under vacuum
and the crude product purified by flash chromatography (silica
gel, petroleum ether/EtOAc 6 : 4, then petroleum ether/EtOAc
1 : 1). A second purification was required (silica gel, petroleum
ether/EtOAc 6 : 4) to afford 6 (12.6 g, 78%) as a white solid. TLC
(silica gel 60 F254, petroleum ether/EtOAc 1 : 1, detection: UV 254
nm): Rf = 0.45; HPLC (system C): Rt = 25.92 min, purity 100%;
1H NMR (CDCl3): d 8.06 (d, J = 8.3 Hz, 2H), 7.40 (m, 5H), 6.96
(d, J = 8.3 Hz, 2H), 5.36 (s, 2H), 4.30 (dd, J = 11.2 Hz, J = 3.0 Hz,
1H), 3.97 (dd, J = 11.2 Hz, J = 3.0 Hz, 1H), 3.37 (m, 1H), 2.92
(dd, J = 4.9 Hz, J = 2.6 Hz, 1H) and 2.77 (dd, J = 4.9 Hz, J =
2.6 Hz, 1H). 13C NMR (CDCl3): d 166, 162, 137, 132, 129, 128,
Resin-anchored oxytocin peptide
In a vessel suitable for SPPS, Fmoc amino acid (2.4 mmol),
N-hydroxybenzotriazole (HOBt) (0.37 g, 2.4 mmol) and N,N¢-
diisopropylcarbodiimide (DIC) (0.38 mL, 2.4 mmol) were added
to a suspension of Rink amide NovaGelTM resin (1.0 g, 0.6 mmol)
in N,N-dimethylacetamide (DMAC) (15 mL). The mixture
was shaken for 3 h at room temperature, the liquid flushed
off and the resin washed with DMAC (5 ¥ 45 mL). The resin
was then shaken with 50% morpholine in DMAC (15 mL) for
15 min, the liquid flushed off and fresh 50% morpholine in
DMAC (15 mL) added. The suspension was shaken for 20 min
more, the liquid flushed off and the resin washed with DMAC
(5 ¥ 15 mL). This procedure was applied sequentially using the
following amino acids: N-a-Fmoc-glycine, N-a-Fmoc-N-e-1-(4,4-
dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine,
3814 | Org. Biomol. Chem., 2009, 7, 3810–3816
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N-a-Fmoc-L-proline, N-a-Fmoc-S-trityl-L-cysteine, N-a-Fmoc-
N-b-trityl-L-asparagine, N-a-Fmoc-N-g-trityl-L-glutamine, N-a-
Fmoc-L-isoleucine and N-a-Fmoc-O-tert-butyl-L-tyrosine. In the
last coupling reaction, N-a-Boc-S-trityl-L-cysteine (1.12 g; 2.4
mmol), HOBt (0.37 g, 2.4 mmol) and DIC (0.38 mL, 2.4 mmol)
were added to the resin in DMAC (15 mL). The mixture was then
shaken for 3 h at room temperature, the liquid flushed off and the
resin washed with DMAC (5 ¥ 15 mL) then CH2Cl2 (5 ¥ 15 mL)
and finally vacuum-dried. The resin-anchored oxytocin peptide
was then used in subsequent steps without further purification.
6.9%; purity according to HPLC system E was 98%, Rt = 6.07
min). MS (ESI+): calc. for [C59H93N17O19S2+ H]+ 1408.63, found
1409; calc. for [C59H93N17O19S2+Na]+ 1430.62, found 1431.0.
Labelling test
The labelling test with 111InCl3 was performed by incubating 5 mg
of conjugate with 500 mCi of 111In(III) at 90 ◦C for 30 min in
0.1 M sodium acetate buffer (pH 5) in the presence of gentisic
acid (0.26 M) as a radical scavenger. The labelling percentage
was determined both by the Sep-Pak method and by FPLC. A
small amount of EDTA solution was added to the incubation
medium in order to complex excess 111In(III). The sample was
loaded onto Sep-Pak and eluted first with 0.1 M sodium acetate to
collect a fraction containing the complex [111InEDTA]- (F1), then
with MeOH to collect a second fraction containing the labelled
oxytocin conjugate (F2). The labelling percentage was calculated
as follows:
8-L-[N6-[4-[2-Hydroxy-3-[4,7,10-tris(carboxymethyl)-1,4,7,10-
tetrazacyclododec-1-yl]propoxy]benzoyl]lysine]oxytocin (9). The
resin-anchored oxytocin peptide (0.5 g, 0.3 mmol) was shaken with
10% hydrazine in DMF (7 mL) for 15 min, the liquid flushed off
and fresh 10% hydrazine in DMF (7 mL) added. The suspension
was stirred for another 20 min, the liquid was then flushed off
and the resin washed with DMF (5 ¥ 7 mL). Bifunctional chelate
8 (0.9 g, 1.2 mmol), HOBt (0.2 g, 1.2 mmol), DIC (0.2 mL, 1.2
mmol), N-ethyldiisopropylamine (0.4 mL, 2.4 mmol) and DMAC
(7 mL) were added to the resin. The mixture was shaken for 40 h at
room temperature, the liquid flushed off and the resin washed with
DMAC (5 ¥ 7 mL), CH2Cl2 (5 ¥ 7 mL) and vacuum dried. The
resin was shaken in a flask with “reagent B” (25 mL) for 4.5 h; the
mixture was then filtered and the filtrate evaporated under reduced
pressure. The resulting oily crude product was treated with Et2O
(5 mL) to give a precipitate that was collected by centrifugation,
washed with Et2O (4 ¥ 20 mL) and vacuum dried. The crude
product was dissolved in H2O (2 L) and DMSO (45 mL), the pH
adjusted to 8.5 by the addition of 25% aq. NH4OH and the solution
stirred for 48 h at room temperature; it was then concentrated and
purified by preparative HPLC (system F). The fractions containing
the product were collected and lyophilised to give 9 as a white
fluffy solid (66 mg, 7%; purity according to HPLC system D was
>99%; Rt = 6.67 min). MS (ESI+): calc. for [C67H101N17O21S2+H]+
1544.69, found 1544.8.
F2 activity
Labelling percentage =
(F1 + F2 )activities
Acknowledgements
We thank Dr Marco Chinol (Istituto Europeo di Oncologia) for
helping with labelling tests.
References
1 M. Woods and A. D. Sherry, Chim. Oggi, 2005, 23, 31.
2 S. Liu, Chem. Soc. Rev., 2004, 33, 445.
3 J. Fichna and A. Janecka, Bioconjugate Chem., 2003, 14, 3.
4 S. Liu and D. S. Edwards, Bioconjugate Chem., 2001, 12, 7.
5 D. E. Milenic, E. D. Brady and M. W. Brechbiel, Nat. Rev. Drug
Discovery, 2004, 488.
6 R. E. Weiner and M. L. Thakur, Appl. Radiat. Isot., 2002, 57, 749.
7 M. Langer and A. G. Beck-Sickinger, Curr. Med. Chem.: Anti-Cancer
Agents, 2001, 1, 71.
8-L-[N 6-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetrazacyclo-
dodec-1-yl]acetyl]lysine]-oxytocin (10). The resin-anchored oxy-
tocin peptide, obtained as described above (1.0 g, 0.6 mmol), was
shaken with 10% hydrazine in DMF (15 mL) for 15 min, the liquid
flushed off and fresh 10% hydrazine in DMF (15 mL) added.
The suspension was stirred for another 20 min, the liquid was
then flushed off and the resin washed with DMF (5 ¥ 15 mL).
DOTA tris-tert-butyl ester (1.56 g, 2.4 mmol), HOBt (0.37 g,
2.4 mmol), DIC (0.38 mL, 2.4 mmol), N-ethyldiisopropylamine
(0.82 mL, 4.8 mmol) and DMAC (15 mL) were added to the
resin. The mixture was shaken for 24 h at room temperature, the
liquid flushed off and the resin washed with DMAC (2 ¥ 15 mL),
CH2Cl2 (5 ¥ 15 mL) and vacuum dried. The resin was shaken in
a flask with “reagent B” (25 mL) for 4 h; it was then filtered and
the filtrate evaporated under reduced pressure. The resulting oily
crude product was treated with Et2O (5 mL) to give a precipitate
that was collected by centrifugation, washed with Et2O (5 ¥ 5 mL)
and vacuum-dried. The crude product was dissolved in H2O (8 L)
and DMSO (420 mL), the pH adjusted to 8.5 by the addition
of 25% aq. NH4OH and the solution stirred for 48 h at room
temperature; it was then concentrated and purified by preparative
HPLC (system G). The fractions containing the product were
collected and lyophilised to give 10 as a white fluffy solid (59 mg,
8 A. Heppeler, S. Froidevaux, A. N. Eberle and H. R. Maecke, Curr.
Med. Chem., 2000, 7, 971.
9 Z. Zhang, S. A. Nair and T. J. McMurry, Curr. Med. Chem., 2005, 12,
751.
10 M. Woods, Z. Kovacs and A. D. Sherry, J. Supramol. Chem., 2002,
2, 1.
11 The Lanthanides and Their Interrelations with Biosystems, Metal Ions
in Biological Systems, ed. A. Sigel and H. Sigel, CRC Press, Boca Raton,
FL, 2003, vol. 40.
12 A. E. Martell and R. M. Smith, Critical Stability Constants, Plenum
Press, New York, 1974, vol. 4.
13 M. L. De Leo´n Rodriguez and Z. Kovacs, Bioconjugate Chem., 2008,
19, 391.
14 A. Heppeler, S. Froidevaux, H. R. Maecke, E. Jermann, M. Behe,
P. Powell and M. Hennig, Chem.–Eur. J., 1999, 5, 1974.
15 K. P. Eisenwiener, P. Powell and H. R. Maecke, Bioorg. Med. Chem.
Lett., 2000, 10, 2133.
16 A. Barge, L. Tei, D. Upadhyaya, F. Fedeli, L. Beltrami, R. Stefan`ıa,
S. Aime and G. Cravotto, Org. Biomol. Chem., 2008, 6, 1176.
17 S. Knor, A. Modlinger, T. Poethko, M. Schottelius, H.-J. Wester and
H. Kessler, Chem.–Eur. J., 2007, 13, 6082.
18 S. J. Kline, D. A. Betebenner and D. K. Johnson, Bioconjugate Chem.,
1991, 2, 26.
19 T. J. McMurry, M. W. Brechbiel, K. Kumar and O. A. Gansow,
Bioconjugate Chem., 1992, 3, 108.
20 M. R. Lewis and J. E. Shively, Bioconjugate Chem., 1998, 9, 72.
21 L. Li, S.-W. Tsai, A.-L. Anderson, D. A. Keire, A. A. Raubitschek and
J. E. Shively, Bioconjugate Chem., 2002, 13, 110.
22 M. F. Tweedle, Eur. Radiol., 1997, 7((Suppl. 5)), S225.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 3810–3816 | 3815
©

View Article Online
23 K. Kumar, C. A. Chang, L. C. Francesconi, D. D. Dischino, M. F.
Malley, J. Z. Gougoutas and M. F. Tweedle, Inorg. Chem., 1994, 33,
3567.
J. C. Reubi, N. Raju, E. C. Metcalfe, L. Lattuada, K. E. Linder, R. E.
Swenson, M. F. Tweedle and A. D. Nunn, J. Nucl. Med., 2006, 47, 1144.
32 G. Gimpl and F. Fahrenholz, Physiol. Rev., 2001, 81, 629.
33 P. Cassoni, E. Fulcheri, M. L. Carcangiu, A. Stella, S. Deraglio and
G. Bussolati, J. Pathol., 2000, 190, 470.
24 P. Caravan, J. J. Ellison, T. J. McMurry and R. B. Lauffer, Chem. Rev.,
1999, 99, 2293.
25 S. M. Quadri and H. M. Vriesendorp, Q. J. Nucl. Med., 1998, 42, 250.
26 S. Geninatti Crich, C. Cabella, A. Barge, S. Belfiore, C. Ghirelli, L.
Lattuada, S. Lanzardo, A. Mortillaro, L. Tei, M. Visigalli, G. Forni
and S. Aime, J. Med. Chem., 2006, 49, 4926.
27 Solid-Phase Synthesis: A Practical Guide, ed. S. A. Kates and
F. Albericio, CRC Press, Boca Raton, FL, 2000.
34 Fmoc Solid Phase Peptide Synthesis: A Practical Approach, ed.
W. C. Chan and P. D. White, Oxford University Press, Oxford, 2000.
35 S. R. Chhabra, B. Hothi, D. J. Evans, P. D. White, B. W. Bycroft and
W. C. C h a n , Tetrahedron Lett., 1998, 39, 1603.
36 J. P. Tam, C.-R. Wu, W. Liu and J.-W. Zhang, J. Am. Chem. Soc., 1991,
113, 6657.
28 E. Cappelletti, L. Lattuada, K. Linder, E. Marinelli, P. Nanjappan,
N. Raju, R. Swenson and M. Tweedle, Int. Pat. Appl., WO2004065407,
2004.
29 L. Lattuada, P. Morosini, F. Uggeri, S. Aime, E. Terreno, D. Delli
Castelli, C. Scolastico, L. Manzoni and D. Arosio, Int. Pat. Appl.,
WO2006095234, 2006.
37 M. Neeman, A. A. Gilad, H. Dafni and B. Cohen, J. Magn. Reson.
Imaging, 2007, 25, 1.
38 E. Garanger, D. Boturyn and P. Dumy, Anticancer Agents Med. Chem.,
2007, 7, 552.
39 R. Ranganathan, E. Marinelli, R. Pillai and M. Tweedle, Int. Pat. Appl.,
WO 9527705, 1995.
30 C. J. Smith, W. A. Volkert and T. J. Hoffman, Nucl. Med. Biol., 2003,
40 N. A. Sole and G. Barany, J. Org. Chem., 1992, 57, 5399.
41 K. Kumar, C. A. Chang, L. C. Francesconi, D. D. Dischino, M. F.
Malley, J. Z. Gougoutas and M. F. Tweedle, Inorg. Chem., 1994, 33,
3567.
30, 861.
31 L. E. Lantry, E. Cappelletti, M. E. Maddalena, J. S. Fox, W. Feng,
J. Chen, R. Thomas, S. M. Eaton, N. J. Bogdan, T. Arunachalam,
3816 | Org. Biomol. Chem., 2009, 7, 3810–3816
This journal is
The Royal Society of Chemistry 2009
©