Ga[NO2A-N-(α-amino)propionate] chelates: Synthesis and evaluation as potential tracers for 68Ga PET

Article abstract of DOI:10.1039/c4dt00386a

The availability of commercial ⁶⁸Ge/⁶⁸Ga cyclotron-independent ⁶⁸Ga³⁺ generators is making Positron Emission Tomography (PET) accessible to most hospitals, which is generating a surge of interest in the design and synthesis of bi-functional chelators for Ga³⁺. In this work we introduce the NO2A-N-(α-amino)propionic acid family of chelators based on the triazacyclononane scaffold. Complexation of the parent NO2A-N-(α-amino)propionic acid chelator and of a low molecular weight (model) amide conjugate with Ga³⁺ was studied by ¹H and ⁷¹Ga NMR. The Ga³⁺ chelate of the amide conjugate shows pH-independent N₃O₃ coordination in the pH range 3-10 involving the carboxylate group of the pendant propionate arm in a 6 member chelate. For the Ga[NO2A-N-(α-amino)propionate] chelate, a reversible pH-triggered switch from Ga³⁺ coordination to the carboxylate group to coordination to the amine group of the propionate arm was observed upon pH increase/decrease in the pH range 4-6. This phenomenon can conceivably constitute the basis of a physiological pH sensor. Both complexes are stable in the physiological range. The [⁶⁷Ga][NO2A-N-(α- benzoylamido)propionate] chelate was found to be stable in human serum. Biodistribution studies of the ⁶⁷Ga³⁺-labeled pyrene butyric acid conjugate NO2A-N-(α-pyrenebutanamido)propionic acid revealed that, despite its high lipophilicity and concentration-dependent aggregation properties, the chelate follows mainly renal elimination with very low liver/spleen accumulation and no activity deposition in bones after 24 hours. Facile synthesis of amide conjugates of the NO2A-N-(α-amino)propionic acid chelator, serum stability of the Ga³⁺ chelates and fast renal elimination warrant further evaluation of this novel class of chelators for PET applications. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4dt00386a

Dalton
Transactions
PAPER
Ga[NO2A-N-(α-amino)propionate] chelates: synthesis
and evaluation as potential tracers for 68Ga PET†
Cite this: Dalton Trans., 2014, 43,
8037
Miguel F. Ferreira,a Goretti Pereira,a João P. André,a M. I. M. Prata,b
Paula M. T. Ferreira,a José A. Martins*a and Carlos F. G. C. Geraldesc,d
The availability of commercial 68Ge/68Ga cyclotron-independent 68Ga3+ generators is making Positron
Emission Tomography (PET) accessible to most hospitals, which is generating a surge of interest in the
design and synthesis of bi-functional chelators for Ga3+. In this work we introduce the NO2A-N-
(α-amino)propionic acid family of chelators based on the triazacyclononane scaffold. Complexation of
the parent NO2A-N-(α-amino)propionic acid chelator and of a low molecular weight (model) amide con-
1
jugate with Ga3+ was studied by H and 71Ga NMR. The Ga3+ chelate of the amide conjugate shows pH-
independent N3O3 coordination in the pH range 3–10 involving the carboxylate group of the pendant
propionate arm in a 6 member chelate. For the Ga[NO2A-N-(α-amino)propionate] chelate, a reversible
pH-triggered switch from Ga3+ coordination to the carboxylate group to coordination to the amine
group of the propionate arm was observed upon pH increase/decrease in the pH range 4–6. This
phenomenon can conceivably constitute the basis of a physiological pH sensor. Both complexes are
stable in the physiological range. The [67Ga][NO2A-N-(α-benzoylamido)propionate] chelate was found to
be stable in human serum. Biodistribution studies of the 67Ga3+-labeled pyrene butyric acid conjugate
NO2A-N-(α-pyrenebutanamido)propionic acid revealed that, despite its high lipophilicity and concen-
tration-dependent aggregation properties, the chelate follows mainly renal elimination with very low liver/
spleen accumulation and no activity deposition in bones after 24 hours. Facile synthesis of amide conju-
gates of the NO2A-N-(α-amino)propionic acid chelator, serum stability of the Ga3+ chelates and fast renal
elimination warrant further evaluation of this novel class of chelators for PET applications.
Received 6th February 2014,
Accepted 7th March 2014
DOI: 10.1039/c4dt00386a
www.rsc.org/dalton
for imaging and therapy. Non-functionalised metal chelates
exhibit high hydrophilicity, distribution into the interstitial
Introduction
Endowing thermodynamically stable and kinetically inert compartment, fast renal clearance and featureless biodistribu-
metal chelates with conjugability is the subject of intensive tion profiles. Delivering metal chelates to injury sites, for
research given the plethora of properties offered by metal ions imaging and/or therapeutic purposes, allows reducing the
dose and background signals and minimizes potential
harmful side effects on non-target organs – especially critical
with complexes of metal-radioisotopes.
Positron Emission Tomography (PET) is well established in
clinical diagnosis for functional imaging thanks to its remark-
aCentro de Química (CQ-UM), Universidade do Minho, Campus de Gualtar,
4710-057 Braga, Portugal. E-mail: jmartins@quimica.uminho.pt
bICNAS and IBILI, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra,
Portugal
ably high detection sensitivity, relatively high spatial resolution
and potential to quantify tracer uptake within lesions. 18F, 11C,
13N are the most widely used radionuclides for PET imaging
due to their positron emission properties and short lifetimes.
These radionuclides are covalently incorporated into bio-
molecules (sugars, amino acids, etc.) through chemical syn-
thesis, starting from simple precursor molecules/ions
generated in dedicated cyclotron units.1 Molecules labeled
with 11C and 13N are indistinguishable from the natural abun-
dance stable isotope unlabelled molecules undergoing the
same metabolic pathways. C-2 18F labeled glucose (2-deoxy-2-
[18F]fluoroglucose – FDG) is the most widely used tracer in
cDepartment of Life Sciences, Faculty of Science and Technology, University of
Coimbra, P.O. Box 3046, 3001-401 Coimbra, Portugal
dChemistry Centre, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal
†Electronic supplementary information (ESI) available: 1D proton and 2D COSY
NMR spectrum (600 MHz) of GaL1 at pH 3.0 (Fig. S1); 1D proton and 2D COSY
NMR spectrum (600 MHz) of GaL1 at pH 4.0 (Fig. S2); UV-Vis spectra for the free
ligand L3 and the GaL3 complex (1.0 × 10−5 M) (Fig. S3); Fluorescence spectra
for L3 in non-deoxygenated water over the concentration range 1.0 × 10−7 and
5 × 10−3 mol dm−3 (λexc = 345 nm) (Fig. S4); Optimized structure of GaL3 (Mopac
PM6 COSMO water implicit solvent) (Fig. S5). Computed log P for the GaL2 and
GaL3 complexes after structure optimization with Mopac PM6 COSMO water
implicit solvent (Table S1); Biodistribution, stated as percentage of injected dose
per gram of organ (%ID g−1), of 67GaL3 in Wistar rats at 1 and 24 hours after i.v.
injection (Table S2). See DOI: 10.1039/c4dt00386a
This journal is © The Royal Society of Chemistry 2014
Dalton Trans., 2014, 43, 8037–8047 | 8037

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Dalton Transactions
clinical PET imaging. Following uptake by the glucose trans- higher kinetic lability than the parent Ga(DOTA)− complex, are
porters and phosphorylation, FDG is trapped inside cells as still kinetically inert enough for in vivo PET.12 The peptide con-
FDG-6-P, allowing visualization in vivo of glucose avid cells and jugate [68Ga](DOTATOC) (DOTATOC = DOTA, Tyr3-octreotide) is
tissues. 18F decay allows the resulting 2-[18OH]glucose to used worldwide in hospitals for diagnosis of neuroendocrine
follow the normal glycolytic pathway.2,3 18F, and especially 11C tumors expressing somatostatin receptors.23,24
and 13N-labelled PET tracers, with half-lives of 109.8, 20.3, and
The macrocycle cavity of DOTA-type chelators, optimal for
9.96 minutes, respectively, need to be produced in loco in dedi- Ln3+ ions, is too big for Ga3+ complexation, thus originating
cated cyclotron units, using fast, high yielding synthetic pro- distorted octahedral (N4O2) complexes.10,12,23,24 Good match-
cedures coupled to expeditious purification schemes (operated ing of the macrocycle cavity of NOTA with the ionic radius of
remotely) to avoid substantial loss of activity.1–3
Ga3+ results in Ga(NOTA) complexes exhibiting exceptional
Many metal ion radionuclides (e.g. 68Ga3+ and 64Cu2+) also thermodynamic stability and kinetic inertness.16,17 Moreover,
display positron emission properties suitable for PET NOTA-type chelators display faster Ga3+ chelation kinetics at
imaging.4 Amongst these, 68Ga3+, with 89% positron branch- lower temperatures than DOTA-type chelators.25 NOTA-mono-
ing accompanied by low photon emission (1077 keV, 3.22%) amide conjugates, although amenable through facile synthetic
and a half life of 67.6 minutes, is suitable for labelling mole- routes, are likely to result in complexes exhibiting lower ther-
cules with fast blood clearance.5,6 The commercial availability modynamic stability and higher kinetic lability than their
of 68Ge/68Ga cyclotron-independent 68Ga3+ generators is parent Ga(NOTA) complex, compromising their potential for
making PET imaging available to most hospitals.7 Labeling biological applications.26 Replacing an acetate arm on the
(bio)molecules with metal ion radionuclides requires efficient NOTA scaffold, by an acetate group α-substituted with a reac-
pathways to attach covalently bi-functional chelators (post-con- tive group, endows the NOTA scaffold with conjugability whilst
jugation labeling), or functionalized chelates (pre-conjugation preserving its N3O3 denticity mandatory for stable coordi-
labeling) to biomolecules.4–7 High thermodynamic stability nation with Ga3+. The aspartate and glutamate derivatives of
and kinetic inertness of the metal complexes are absolutely NOTA, NODASA (NODASA = 1,4,7-triazacyclononane-N-succinic-
mandatory for safe and efficient PET imaging.8–10 Transferrin, N′,N″-diacetic acid)27 and NODAGA (NODAGA = 1,4,7-triaza-
the iron transport protein, is the main competitor for Ga3+ cyclononane-N-glutaric-N′,N″-diacetic acid),28 respectively,
in serum given the similar ionic radius and coordination are the gold standards for the synthesis of targeted Ga3+
properties of Fe3+ and Ga3+.11 In addition to stability, fast tracers for PET. More recently, the TRAP (TRAP = 1,4,7-triaza-
complexation, ideally at room temperature, and efficient cyclononane-1,4,7-tris(methylenephosphinic acid)) family of
purification procedures of labeled conjugates have to be taken chelators, generated by replacement of the pendant carboxy-
into account in the design of new chelators for Ga3+ for use as lates by phosphinate groups on the triazacyclononane macro-
PET tracers.12 Moreover, complex formation with Ga3+, cycle scaffold, revealed excellent chelating properties for Ga3+
obtained as 68GaCl3 by elution of 68Ge/68Ga generators with allied to conjugability.29–31 Tris-NODASA/NODAGA32 and tris-
diluted hydrochloric acid, must be carried out at pH values TRAP29 derivatives allow facile preparation of multivalent con-
below 4 (usually in acetate or HEPES buffer) to avoid precipi- jugates with biomolecules such as peptides, exhibiting
tation of Ga3+ as Ga(OH)3.13,14
enhanced avidity (affinity and/or specificity) for cellular recep-
The coordination chemistry of Ga3+ is dominated by its tors. Pendant reactive groups on the ethylenediamine bridges
hard character, preferring hard, charged base donors such as of the NOTA scaffold allow also bioconjugation whilst retaining
carboxylates, phosphonates and phosphinates in an octahedral the high thermodynamic stability and kinetic inertness of the
geometry,10 although it has also been shown to have good Ga(NOTA) complex, although at the expense of difficult and
affinity for thiolates, such as TACN-TM (TACN-TM = 1,4,7-tri- lengthy synthetic pathways.33
azacyclononane-N,N′,N″-tris(mercaptoethyl)).10,15 Poly(amino-
In our efforts to obtain thermodynamically stable and kine-
carboxylates) and poly(aminophosphonates/phosphinates) are tically inert conjugates of metal chelates as potential medical
the most efficacious chelators for Ga3+. Macrocyclic, DOTA- imaging agents, we have reported recently efficient methodo-
type (DOTA = 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetic logies for the synthesis of the DO3A-N-(α-amino)propionate
acid) and NOTA-type (NOTA = 1,4,7-triazacyclononane-1,4,7- chelator34 and for the preparation of amide conjugates.35–37
triacetic acid) chelators form Ga3+ complexes displaying excep- The Gd[DO3A-N-(α-amino)propionate] chelate and its amide
tional thermodynamic stability and kinetic inertness.12,16–18 conjugates combine high thermodynamic stability and kinetic
Ga3+ complexes of linear, DTPA19 (DTPA = diethylenetriamine inertness with (fast) water exchange properties, ideal for attain-
pentaacetic acid) and EDTA-type20 (EDTA = ethylenediamine- ing high relaxivities at (intermediate/high) fields relevant for
tetraacetic acid) chelators were found to be kinetically too magnetic resonance imaging.34–37 In this work we extend the
unstable for in vivo use, despite the high thermodynamic stabi- synthetic methodologies developed for DO3A-N-(α-amino)pro-
lity of its Ga(EDTA)− and Ga(DTPA)2− complexes.21,22 DOTA- pionate chelators to the triazacyclononane scaffold. This
monoamide (octadentate) chelators, developed originally as includes the NO2A-N-(α-amino)propionic acid (L1) chelator
Gd3+ complexing agents for use as Contrast Agents (CA) for and the two amide derivatives NO2A-N-(α-benzoylamido)-
Magnetic Resonance Imaging (MRI), despite forming Ga3+ propionic acid (L2) and NO2A-N-(α-pyrenebutanamido)propionic
complexes exhibiting lower thermodynamic stability and acid (L3) (see Scheme 1). The complexation of the new chela-
8038 | Dalton Trans., 2014, 43, 8037–8047
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Dalton Transactions
Paper
ation of nonane 5 with tert-butyl bromoacetate, followed by
sequential deprotection with hydrochloric acid (4 M) and
Dowex-1X2-OH− resin, and elution with diluted hydrochloric
acid afforded the NO2A-N-(α-amino)propionic acid chelator
(L1) as hydrochloride in (non-optimised) 36% overall yield.
Alkylation of monoalkylated cyclen 5 with ethyl bromoacetate
offers the opportunity to deprotect selectively the amine group
for further conjugation.35 This (direct) conjugation pathway
was not pursued in this work. Instead, we have explored the
indirect pathway for preparing amide conjugates of the NO2A-
N-(α-amido)propionic acid chelator.36 The NO2A-N-(α-benzoyl-
amido)propionic acid chelator (L2) is likely to result in soluble
non-associating chelates; the pyrenyl conjugate NO2A-N-(α-pyr-
enebutanamido)propionic acid chelator (L3) was designed as a
self-assembling, fluorescent chelator, potentially useful for
bimodal PET/fluorescence imaging.37 Michael addition of Boc-
(dehydroamide)-Δ-AlaOMe blocks 3 and 4 introduces directly
the amide (targeting/kinetic modulator) group into the triaza
scaffold. Removing the Boc group at the monoalkylated stage
averts retro-elimination during the second alkylation step. One
step deprotection of the ester protected prochelators 9 and 10
with Dowex-1X2-OH− resin affords the fully deprotected chela-
tors L2 and L3 as hydrochlorides, after elution with aqueous
hydrochloric acid.
Scheme 1 Synthetic pathway for the metal chelators NO2A-N-
(α-amino)propionic acid (L1), NO2A-N-(α-benzoylamido)propionic acid
(L2) and NO2A-N-(α-pyrenebutanamido)propionic acid (L3). (a) K2CO3/
MeCN; (b) tert-butyl bromoacetate, K2CO3/MeCN; (c) i. hydrochloric
acid/EtOH, ii. Dowex-1X2-OH−, elution with diluted hydrochloric acid;
(d) GaCl3·xH2O; (e) i. TFA/CH2Cl2; ii. ethyl bromoacetate, K2CO3/MeCN;
(f) Dowex-1X2-OH−, elution with diluted hydrochloric acid.
Many other amide conjugates are accessible using the expe-
ditious methodologies described in this work.
1H and 71Ga NMR studies
Given the current interest in 68Ga PET, unleashed by the
commercial availability of cyclotron-independent 68Ga
generators,4–7 we carried out a multinuclear NMR study (1H
and 71Ga) of the GaL1 and GaL2 chelates to ascertain the
coordination properties of the novel chelators and their poten-
tial for PET imaging applications.
tors with Ga3+ was studied by multinuclear NMR (1H, 71Ga).
The luminescence properties of the GaL3 complex were also
analysed in order to evaluate its potential as a bimodal PET/
optical imaging agent. Serum stability and biodistribution
studies were also performed to evaluate the potential of the
new chelators as 68Ga3+-labeled PET tracers.
71Ga (38.89%) is a quadrupolar nucleus (I = 3/2) exhibiting
a relatively high receptivity and a wide range of chemical
shifts.38 The quadrupolar nature of its relaxation mechanism
leads to NMR signals whose linewidth is sensitive to the sym-
metry of the coordination environment. Spherical, octahedral,
cubic and tetrahedral symmetries should, in principle, orig-
inate sharp 71Ga NMR signals. This is the case for NOTA-type
triazamacrocyclic complexes, whose predominant coordination
Results and discussion
Synthesis and characterization
In this work we aimed at extending the synthetic methodo- geometry is distorted octahedral, as opposed to DOTA-type
logies
developed
for
DO3A-N-(α-amino)propionate tetraazamacrocyclic complexes like Ga(DOTA)- and its mono-
chelators34–37 to the triazacyclononane scaffold, to explore the amide derivatives, whose low coordination symmetry makes
coordination properties of NO2A-N-(α-amino)propionic acid their 71Ga NMR signals too broad to be detected.12 The chemi-
chelators for Ga3+ as potential tracers for PET imaging cal shift is characteristic of the coordination geometry of the
(Scheme 1).
Michael addition of the Boc2-Δ-AlaOMe (2)34 and Boc-
metal ion.39,40
The pH dependence of the 1H (Fig. 3) and 71Ga NMR
(amide)-Δ-AlaOMe blocks 336 and 437 to triazacyclononane (1) spectra (Fig. 1) for the GaL1 and GaL2 complexes was studied
proceeds smoothly in acetonitrile at room temperature, using in D2O to probe the coordination environment of the Ga3+
K2CO3 as a base, in 3–4 hours, as reported before for the cyclen metal ion.
analogue. An excess of triazacyclononane, 1.5 molar equi-
Table 1 summarizes the 71Ga NMR chemical shifts and line-
valents, affords the monoalkylated nonanes as the main reaction widths for the species observed in solution for the GaL1 and
products. Small amounts of bis- and tris-alkylated derivatives GaL2 complexes studied in this work, compared with other
are easily removed by flash chromatography. Further N-alkyl- triazamacrocyclic complexes reported in the literature (Fig. 2).
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Fig. 1 pH dependence of the 71Ga NMR spectra for: (A) the GaL1 and (B) GaL2 complexes (400 MHz, 20 mM in D2O, 25 °C).
Table 1 71Ga NMR chemical shifts and linewidths for the GaL1 and GaL2
complexes studied in this work, compared with other triazamacrocyclic
complexes reported in the literature40
Complex
δ (ppm) Δν1/2 (Hz) Coordination
a
GaL1
+141
+186
+140
+171
900
550
900
235
283
9085
279
4818
434
215
154
200
186
162
N3O3
N4O2
N3O3
N3O3
N3O3
N4O2
N3O3
N4O2
N3O3
N3O3
N3O3
N3O3
N3O3
N3O3
a
GaL2
Ga(NOTA)b
Ga(NO2A-N-methylacetamide)c +171
Ga(NO2A-N-benzylacetamide)c
+175
+170
+174
+110
+136
+132
+134
+138
+141
Ga(NOTP)d
Ga(NOTMP)e
Ga(TRAP-H)f
Fig. 2 Structure of triazacyclononane ligands compared in Table 1 and
discussed in the manuscript: NOTP (1,4,7-triazacyclononane-N,N’,N’’-
tris(methylenephosphonic) acid); NOTMP (1,4,7-triazacyclononane-N,N’,
N’’-tris(methylene(methylphosphinate))); TRAP-H (1,4,7-triazacyclo-
nonane-1,4,7-tris(methylenephosphinic) acid).
a This work. b Ref. 17,41. c Ref. 42. d Ref. 43. e Ref. 44. f Ref. 30.
for the amide complex GaL2, the coordination polyhedron is
The number and population of the species present over the likely to be represented by a distorted octahedron involving
pH range 3–11, as well as their chemical shifts and linewidths, the carboxylate group of the propionate arm to form a six
are quite different for the GaL1 and GaL2 complexes.
member chelate (coordination polyhedron N3O3). Although
The GaL2 complex displays only one 71Ga NMR signal in coordination through the amide nitrogen, forming a five
the pH range 3–9: a singlet at δ = 140 ppm with a relatively member chelate, is also possible, the preference of the hard
large linewidth, Δν1/2 = 900 Hz. Above pH 9.0, hydroxide cata- Ga3+ ion for the carboxylate group is likely to compensate for
lyzed demetallation leads to the formation of the [Ga(OH)4]− the energy cost of forming a six member chelate.42 The GaL1
species characterized by a sharp signal at δ = 224 ppm.40 In chelate seems to undergo the same chelation scheme (N3O3)
contrast, the 71Ga NMR spectrum of the GaL1 chelate is pH as the amide complex at low pH, presumably due to the un-
dependent. At pH 3.0 there is only one signal with a chemical availability of the protonated amine group for binding. At
shift (141 ppm) and width (900 Hz) identical to that displayed higher pH (6–11) the formation of a five member chelate with
by the GaL2 complex. A new sharper (Δν1/2 = 550 Hz) signal at the deprotonated amine group (coordination polyhedron
δ = 186 ppm becomes apparent at pH 4.1. The intensity of this N4O2) seems to be more favorable than the formation of a six
signal increases, at the expense of the signal at δ = 141 ppm, member chelate with the harder carboxylate group. The N4O2
when the pH increases, eventually becoming the only signal in chelation mode for the GaL1 chelate (all five member chelates)
the pH range 6–10. At pH 11, the [Ga(OH)4]− species is also is likely to generate a coordination polyhedron of higher sym-
present. This process is fully reversible, reproducing the same metry, similar to Ga(NOTA), than the N3O3 geometry involving
features upon pH decrease. These results strongly suggest that a six member chelate – thus, the sharper 71Ga NMR signal and
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higher chemical shift of the N4O2 isomer of GaL1 at pH 6–11.
For the GaL3 complex at 20 mM concentration, the 71Ga NMR
spectrum becomes too broad to be measured, reflecting prova-
bly the micellar state of the complex.45 Both GaL1 and GaL2
complexes are stable in the physiological pH range undergoing
hydroxide catalyzed demetallation above pH 10 and 9, respecti-
vely. This is apparent in the 71Ga NMR spectra by the dis-
appearance of the signals assigned to the complexes and by the
appearance of the sharp signal at δ = 224 ppm, assigned to the
[Ga(OH)4]− ion. All known Ga3+ complexes undergo hydroxide
catalyzed demetallation in the alkaline pH region.40 In this
respect, the GaL1 complex seems to be more stable than the
amide conjugate GaL2 chelate, requiring a higher pH for com-
plete demetallation. This can be ascribed to the preference for
N4O2 coordination of the GaL1 complex at high pH, compared
to the N3O3 coordination of GaL2. A similar isomerization
Fig. 3 pH dependence of the proton NMR resonances of the CH
protons of the propionate side arm of the GaL1 and GaL2 complexes
process involving coordination of the Ga3+ ion to the nitrogen (400 MHz, 20 mM in D2O, 25 °C).
or oxygen atom of the amide linkage (five member chelates)
has been observed for the Ga(NO2A-N-benzylacetamide) and
Ga(NO2A-N-methylacetamide) complexes (Fig. 2 and Table 1),
7.40 ppm with a 2 : 2 : 1 intensity ratio, corresponding to the
both in the solid state and in aqueous solution. At low pH the
N3O3 coordination predominates over N4O2 coordination.42 As
can be seen in Table 1, the two isomers of the Ga(NO2A-N-
benzylacetamide) and Ga(NO2A-N-methylacetamide), involving
in both cases five member chelates, show much smaller shift
differences and are much broader than the isomers found
in the present study for GaL1. The phosphonate complex
Ga(NOTP)43 and some phosphinate complexes, like
Ga(NOTMP),44 lead to the presence of only one species in solu-
tion. In all the NOTA-type complexes with N3O3 coordination,
the Ga3+ ion is placed between twisted trigonal O3 and N3
planes, an arrangement which leads to chiral complexes. This
is caused by the combination of the conformation of the
chelate rings in the macrocyclic moiety (δδδ/λλλ) with the heli-
city of the coordinated pendant arms (Δ/Λ), which leads to two
diastereoisomeric pairs Δδδδ/Λλλλ and Λδδδ/Δλλλ. In the NOTA
derivatives bearing three phosphinate pendant arms, those
chiralities are combined with the R/S chirality of the phos-
phorus atom upon metal coordination, leading to four poss-
ible diastereomeric pairs, Λδδδ –RRR/Δλλλ–SSS, Λδδδ–RRS/Δλλλ–
SSR, Λδδδ–RSS/Δλλλ–SRR and Λδδδ–SSS/Δλλλ–RRR. In the Ga3+
phosphinate complexes with alkyl substituents (CH3 in Ga-
(NOTMP)44 or CH2OH in Ga(TRAP-OH),30 only the Λδδδ–RRR/
Δλλλ–SSS diastereoisomer is observed in solution, leading to a
single 71Ga signal, while for more electron-withdrawing substi-
tuents, like H in Ga(TRAP-H),30 the full mixture of possible
isomers is observed in solution as a set of four narrow reson-
ances with very small shift differences (Table 1).
ortho, para and meta CH protons. The most informative reso-
nances correspond to the NCH2CH protons of the substituted
propionate arm of the complexes, in the form of a character-
istic ABX pattern, with the CH proton resonances outside the
envelope of other protons (Fig. S1 and S2†). The AB patterns of
the CH2 signal coupled to the CH group (NCH2CH) could be
traced to the COSY spectra of both complexes (Fig. S1 and
S2†). In the case of GaL2 the CH multiplet has a single com-
ponent at 5.05 ppm (Fig. 3), in accordance with the single 71Ga
resonance at +140 ppm, corresponding to N3O3 coordination
(Fig. 1). In the case of GaL1 the CH proton signals are pH-
dependent, in parallel with what was observed for the 71Ga
NMR spectra (Fig. 3). Whereas at pH 3.3, it consists of a single
multiplet (doublet of triplets) at 4.47 ppm (labeled I), corres-
ponding to the single 71Ga resonance at 141 ppm (N3O3 coordi-
nation isomer), at pH 4.1 there are two CH multiplets at 4.50
(I) and 4.38 ppm (II), corresponding to the 71Ga signals at 141
and 186 ppm, assigned to the N3O3 and N4O2 isomers, respect-
ively. The relative intensity of the N4O2 signals (4.38 ppm and
186 ppm) increases with pH in relation to the N3O3 signals
(4.50 and 141 ppm) and at pH 6.1 only the 4.38 ppm signal is
present. The corresponding CH2 multiplets are observed
between 3.60 and 3.20 ppm (Fig. S1 and S2†). Therefore, the
proton NMR spectra confirm the isomer information provided
by the 71Ga NMR spectra.
Fluorescence studies
The 1D 1H and 2D COSY and NOESY spectra of the GaL1 The GaL3 complex was designed to have properties compatible
complex solutions were also obtained at 25 °C in the pH range with a bimodal PET/fluorescence imaging agent. The lipophilic
3.0–9.0, and at pH 3.0–5.5 for GaL2. The proton resonance pyrene moiety was selected due to its special aggregation-
signals from the macrocyclic backbones were only tentatively sensitive fluorescence properties.37,46 The absorption (Fig. S3†)
assigned due to superimposed multiplet signals in the and the fluorescence emission properties (Fig. S4†) of the free
3.7–2.8 ppm range (Fig. S1 and S2†). The NCH2CO2H acetate ligand L3 and of the GaL3 complex (Fig. 4) were studied in
arm protons give a multiplet at 3.85 ppm, while the phenyl non-deoxygenated water (pH 7.0) by UV-Vis and steady-state
group of GaL2 originates three multiplets at 7.70, 7.45 and fluorescence spectroscopy (Table 2).
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Dalton Transactions
equal to the cmc value) are present in solution. Increasing the
ligand or chelate concentration leads to an increase of the
number of micelles, enhancing excimer emission.
In vitro stability and biodistribution studies
The kinetic stability of the [67Ga]L2 chelate towards in vivo
transchelation was assessed by incubating the 67Ga3+-labeled
chelate with fresh human serum, followed by precipitation of
the protein fraction and measurement of the activity in the
pellet and in the soluble fraction (Fig. 5).
The percentage of total activity in the protein pellet reached
immediately a value of 10% after incubating the complex
with serum. This value remained constant over 1 hour incu-
bation, revealing early association of the complex with blood
proteins, presumably involving the lipophilic benzoyl moiety
(Log Pcalculated(GaL2) = 0.69, see ESI†). Radio-TLC analysis of
the soluble fraction after 1 hour incubation revealed that the
complex remains intact.
Fig. 4 Normalized fluorescence spectra for GaL3 in non-deoxygenated
water over the concentration range 5 × 10−7–5 × 10−3 mol dm−3 (λexc
=
345 nm).
Table 2 Quantum yields (ΦF) for the ligand L3 and GaL3 complex (λex
345 nm) in non-deoxygenated water
Biodistribution profiles were obtained for the [67Ga]L3
labelled complex at 1 and 24 hour post-injection in Wistar rats
(Fig. 6, Table S1†).
a,b
ΦF
L3
GaL3
At 1 hour post-injection the activity is manly located in the
kidneys, liver and blood and to a lesser extent in the lungs and
intestines. The high kidney uptake, compared to liver, indi-
cates that the main elimination pathway is renal. The relatively
high activity in the blood at 1 hour post injection suggests
association of the complex with plasma proteins, probably
0.20
0.30
a Relative to anthracene in ethanol (Φ = 0.27). b Ligand and complex at
1.0 × 10−6 M concentration, non-deoxygenated solutions.
The absorption spectra of L3 and GaL3 (1 × 10−5 M, pH 7.0)
exhibit bands in the wavelength range 300–350 nm character-
istic of pyrene intraligand π–π* transitions (Fig. S3†).47 The
steady state fluorescence spectra of L3 (Fig. S4†) and GaL3
(Fig. 4) at low micromolar concentrations (1 × 10−6 M, λex
=
345 nm) display the typical vibronically structured band, attrib-
uted to the pyrene monomer (1π–π* transitions).37,48 The fluo-
rescence quantum yields for L3 and for the pyrene-centred
emission in GaL3 are of the same order of magnitude as those
reported for other pyrene conjugates.37,47,48 Complex for-
mation with diamagnetic Ga3+ enhances slightly the fluo-
rescence quantum yield of L3. The concentration dependence
of the steady state fluorescence properties of L3 (Fig. S4†) and
GaL3 (Fig. 4) indicates that both, ligands and complex,
undergo self-assembly, presumably into micelle-type struc-
tures, in aqueous solution.
Fig. 5 Percentage of activity in the protein and in the soluble fraction
at different incubation times of [67Ga]L2 with human serum.
Increasing the concentration of L3 and GaL3 triggers a
gradual switch from the characteristic structured pyrene
monomer spectrum to the broad featureless typical excimer
(excited dimer) spectrum, indicating self-assembly in solution,
presumably in the form of micelle-type structures.37 Attempts
to calculate the critical micelle concentration (cmc) for L3 and
GaL3, by fitting the concentration dependence of the ratio of
fluorescence emission intensities for the excimer (490 nm) and
for the monomer (377 nm) (IE/IM) to a sigmoidal curve model,
proved unsuccessful.37,49,50 Nonetheless, the aggregation-
sensitive nature of the fluorescence emission is clearly observable.
Above the cmc, aggregates and monomers (at a concentration
Fig. 6 Biodistribution profiles, presented as % injected activity per gram
of organ or tissue, for the [67Ga]L3 complex at 1 and 24 hours post-
injection. Results are the average of 4 animals.
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Paper
albumin or lipoproteins, presumably through the lipophilic Reactions were monitored by TLC on a Kieselgel 60 F254
pyrene moiety (Log Pcalculated.(GaL3) = 3.31, see ESI†).37 The (Merck) on an aluminum support, with detection by examin-
very low spleen and lung uptake suggests that in the blood the ation under UV light (254 nm and 365 nm), by adsorption of
complex is not aggregated. A motivating factor for performing iodine vapor and by spraying with ninhydrin. Flash chromato-
the biodistribution study was to ascertain potential brain graphy was performed on a Kieselgel 60 (Merck, mesh
uptake of GaL3. Biodistribution studies of the analogous 230–400). Ion exchange chromatography was performed on
[
153Sm][DO3A-N-(α-pyrenebutanamido)propionate] complex sug- Dowex-1X2-OH− resin (Sigma Aldrich). The resin was pur-
gested a low, although not negligible, activity uptake at the chased as the Cl− form and converted to the OH− form by the
brain-cerebellum at 1 hour pi.37 The higher lipophilicity of standard procedure. The relevant fractions from chromato-
GaL3 (overall neutral complex), compared to its DOTA analogue graphy were pooled and concentrated under reduced pressure,
Sm[DO3A-N-(α-pyrenebutanamido)propionate] complex (overall T < 40 °C. 1H and 13C NMR spectra (assigned by 2D DQF-COSY
charge −1), could conceivably result in higher brain uptake. and HMQC techniques) were run on a Varian Unity Plus 300
The current study excludes brain uptake of [67Ga]L3. Most and a Bruker Avance-3 400 Plus NMR spectrometer, operating
importantly, no significant activity remains in the liver and at 299.938 MHz and 75.428 MHz, and 400.13 MHz and
spleen and there is no activity deposition in the bones after 100.61 MHz, for 1H and 13C, respectively. 1H 1D and 2D
24 hours, indicating that complex demetallation is not occur- DQF-COSY and NOESY spectra were also run on a Varian
ring in vivo.
VNMRS 600 at 599.72 MHz. Chemical shifts (δ) are given in
ppm relative to the CDCl3 solvent (1H, δ 7.27; 13C 77.36) as an
internal standard. For 1H and 13C NMR spectra recorded in
D2O, chemical shifts (δ) are given in ppm, respectively, relative
to TSP as an internal reference (1H, δ 0.0) and tert-butanol as
Conclusions
In this work we extend the synthetic pathways, reported pre- an external reference (13C, CH3 δ 30.29). 71Ga NMR spectra
viously for the DO3A-N-(α-amino)propionate family of chela- were recorded at 122.026 MHz on a Bruker Avance-3 400 Plus
tors, to the triazacyclononane scaffold. A reversible pH- spectrometer, using the signal of [Ga(H2O)6]3+ present in a
triggered switch from Ga3+ coordination to the carboxylate 0.1 M Ga(NO3)3 solution in D2O, at 0 ppm as an external refer-
group of the substituted propionate arm (at pH values below ence. pH measurements were performed on a pH meter Crison
4.0) to amine coordination (at pH values above 6.0) was un- micro TT 2050 with an electrode Mettler Toledo InLab 422.
covered by 1H and 71Ga NMR studies of the Ga[NO2A-N- For D2O solutions, the isotopic correction pD = pH + 0.4
(α-amino)propionate] complex. The Ga3+ chelate of the benzoyl- was done.51 Mass spectrometry was performed at CACTI, Vigo,
amide conjugate displays pH-independent Ga3+ complexation Spain.
to the carboxylate of the amide-substituted propionate arm in
Synthetic procedures
the pH range 3–11. Both complexes are stable in the physio-
logical pH range. Moreover, the [67Ga][NO2A-N-(α-benzoyl-
amido)propionate] chelate is also stable in human serum. The
pyrenebutyric acid conjugate, Ga[NO2A-N-(α-pyrenebutan-
Preparation of monoalkylated nonanes 5, 6 and 7
Synthesis of bis(tert-butoxycarbonyl)amino-methoxycarbonyl-
ethyl-1,4,7-triaza cyclononane
–
monoalkylated nonane
amido)propionate] chelate, was designed as
bimodal PET/fluorescence imaging agent with aggregation-
sensitive fluorescence properties.
a potential
(5). Boc2-Δ-Ala-OMe (2) (158 mg, 0.51 mmol) was added in
one portion to a suspension containing 1,4,7-triazacyclo-
nonane (98 mg, 0.76 mmol) and K2CO3 (279 mg, 2.0 mmol) in
MeCN (20 mL). The suspension was vigorously stirred at room
temperature for 2 hours. The suspended solid was removed by
filtration and the solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography
(100% CH2Cl2 → CH2Cl2–EtOH–NH4OH–H2O (50 : 50 : 1 : 1)) to
afford compound 5 (120 mg, 55%). 1H NMR (300 MHz, CDCl3):
δ = 1.51 (s, 18 H, Boc), 2.58–2.80 (m, 12 H, N(CH2)2N), 3.65
(dd, J = 14.1 and 8.4 H z, 1 H, ABX), 3.70 (dd, J = 14.1 and 4.5
Hz, 1 H, ABX), 3.71 (s, 3 H, C(O)OMe), 5.01 (dd, J = 8.4 and 4.5
Hz, 1 H, ABX).13C NMR (75.4 MHz, CDCl3): 28.02 (C(CH3)3),
46.45 (CH2), 46.83 (CH2), 52.16 (C(O)OCH3), 53.16 (CH2), 56.52
(CH2), 56.67 (CH), 83.34 (C(CH3)3), 152.28 (C(O), carbamate),
170.73 (C(O), ester). HRMS (ESI): m/z: calcd for C20H39N4O6,
[M + H]+: 431.2864, found: 431.2859.
The facile synthesis of conjugates of the NO2A-N-(α-amino)-
propionic acid chelator, together with the serum stability of
their Ga3+ chelates and fast renal elimination of the lipophilic
pyrene conjugate, without any liver/spleen retention and
activity deposition in the bones, warrants further evaluation of
this novel class of chelators as potential tracers for PET.
The reversible pH-triggered coordination switch demonstrated
for the Ga[NO2A-N-(α-amino)propionate] complex can concei-
vably be translated into a method for pH measurement in vivo
by 71Ga NMR.
Experimental
Materials and methods
Synthesis of N-tert-butoxycarbonylbenzamido-methoxycarbonyl-
Chemicals were purchased from Sigma-Aldrich and used
ethyl-1,4,7-triaza cyclononane
–
monoalkylated nonane
without further purification. Analytical grade solvents were (6). (Benzyl)Boc-Δ-AlaOMe (3) (385 mg, 1.23 mmol) was added
used without further purification, unless otherwise specified. in one portion to a suspension containing 1,4,7-triazacyclo-
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Dalton Transactions
nonane (242 mg, 1.87 mmol) and K2CO3 (691 mg, 4.5 mmol) 1H NMR (300 MHz, CDCl3): δ = 1.44 (s, 18 H, C(CH3)3, Boc),
in MeCN (70 mL). The suspension was vigorously stirred at 1.65 (s, 18 H, C(CH3)3, ester), 2.75–3.78 (m(br) 12 H,
room temperature for 3 hours. The suspended solid was N(CH2)2N), 3.03 (t, J = 4.2 Hz, 1 H, ABX), 3.20 (t, J = 4.2 Hz, 1 H,
removed by filtration and the solvent was evaporated under ABX), 3.73 (s, 4 H, NCH2C(O)), 3.78 (s, 3 H, C(O)OMe), 5.01 (t,
reduced pressure. The residue was purified by flash chromato- J = 4.2 Hz 1 H, ABX).13C NMR (75.4 MHz, CDCl3): 28.02
t
graphy (100% CH2Cl2
→
CH2Cl2–EtOH–NH4OH–H2O (C(CH3)3, Boc and Bu ester), 44.53 (3 C, NCHCH2, CH2), 50.80
(70 : 30 : 1 : 1)) to afford monoalkylated nonane 6 as a viscous (2 C, CH2), 53.16 (2 C, CH2), 52.33 (3 C, OCH3, 2 × CH2), 56.52
1
light yellow oil (364 mg, 59%). H NMR (300 MHz, CDCl3): δ = (CH), 81.00 (2 C, C(CH3)3), 83.50 (2 C, C(CH3)3), 150.59 (1 C,
1.47 (s, 9 H, C(CH3)3), 2.50–2.90 (m, 12 H, N(CH2)2N), 3.01 (dd, C(O)), 151.98 (1 C, C(O), carbamate), 163.95 (1 C, C(O)OCH3),
J = 14.1 and 6.0 Hz, 1 H, ABX), 3.49 (dd, J = 14.4 and 5.1 Hz, 170.98 (2 C, C(O)). HRMS (ESI): m/z: calcd for C32H59N4O10,
1 H, ABX), 3.76 (s, 3 H, C(O)OMe), 5.25 (t, J = 6.0 and 5.1 Hz, [M + H]+, 659.4226, found: 659.4218.
1 H, ABX), 7.30–7.70 (5 H, m, Ar). 13C NMR (75.4 MHz, CDCl3):
Synthesis of benzamido-methoxycarbonylethyl-4,7-bis-(ethoxy-
carbonylmethyl)-1,4,7-triaza cyclononane – tris-alkylated nonane
9. A solution of monoalkylated compound (340 mg,
δ = 27.27 (C(CH3)3), 44.77 (CH2), 45.83 (CH2), 46.70 (CH2),
51.27 (CH2), 52.51 (C(O)OCH3), 55.61 (NCH2CH), 56.08 (CH),
83.88 (C(CH3)3), 127.87 (Ar), 127.96 (Ar), 131.27 (Ar), 137.02
(Ar), 152.72 (C(O), carbamate), 170.57 (C(O), amide), 172.62
(C(O), ester). HRMS (ESI): m/z: calcd for C22H35N4O5, [M + H]+:
435.2602, found: 435.2601.
6
0.78 mmol) in trifluoroacetic acid in dichloromethane (60%,
25 mL) was stirred overnight at room temperature. The solvent
was evaporated at reduced pressure. The residue was re-
dissolved in dichloromethane and the solvent was evaporated.
This procedure was repeated several times to give a light yellow
thick oil. 1H NMR spectroscopy (CDCl3) revealed the dis-
Synthesis of methoxycarbonyl-(2-(N-t-butoxycarbonyl-4-pyrene-
butanamido))-ethyl-4,7-bis-(ethoxycarbonylmethyl)-1,4,7-triazacyclo-
nonane – monoalkylated 7. (Pyrenyl)Boc-Δ-AlaOMe (4) (1.01 g, appearance of the signals assigned to the Boc group in the pre-
2.14 mmol) was added in one portion to a suspension contain- cursor compound. The oil (0.78 mmol, assuming quantitative
ing 1,4,7-triazacyclononane (0.43 g, 3.33 mmol) and K2CO3 deprotection) was dissolved in MeCN (30 mL), K2CO3 (1.0 g,
(1.76 g, 12.7 mmol) in MeCN (40 mL). The suspension was vig- 7.8 mmol) was added and the suspension was vigorously
orously stirred at room temperature for 5 hours. The sus- stirred at room temperature for 15 minutes. Ethyl bromo-
pended solid was removed by filtration and the solvent was acetate (0.21 mL, 1.88 mmol) was added and the suspension
evaporated under reduced pressure. The residue was purified was further stirred at room temperature for 2.5 hours. The sus-
by flash chromatography (100% CH2Cl2 → CH2Cl2–EtOH– pended solid was removed by filtration, the solvent was evapor-
NH4OH–H2O (70 : 30 : 1 : 1)) to afford monoalkylated nonane 7 ated under reduced pressure and the residue was purified by
1
as a viscous light yellow oil (0.76 g, 59%). H NMR (300 MHz, flash chromatography (100% CH2Cl2 → CH2Cl2–EtOH (7 : 3)) to
1
CDCl3): δ = 1.42 (s, 9 H, C(CH3)3), 2.21 (m, 2 H, NHC(O)- afford compound 9 (355 mg, 37%) as a white foam. H NMR
CH2CH2), 2.64 (m, 8 H, 2 × N(CH2)2N), 2.90 (m, 1 H, ABX), 3.12 (300 MHz, CDCl3): δ = 1.22 (m, 6 H, C(O)OCH2CH3), 2.2–3.60
(m, 6 H, N(CH2)2N and NHC(O)CH2), 3.38–3.50 (m, 3 H, NHC- (broad multiplet, 18 H, N(CH2)2N, NCH2C(O) and NCH2CH),
(O)CH2CH2CH2 and ABX), 3.43 (m, 2 H, N(CH2)2N), 3.70 (s, 3.71 (s, 3 H, C(O)OCH3), 4.11 (t, J = 7.5 Hz, C(O)OCH2CH3),
3 H, C(O)OMe), 5.44 (t, J = 6.9 and 4.8 Hz, 1 H, ABX), 7.95–8.40 5.14 (m (br), 1 H, NCH2CH), 7.44–8.03 (5H, m, Ar). 13C NMR
(9 H, m, Ar). 13C NMR (75.4 MHz, CDCl3): δ = 27.03 (CH2), (75.4 MHz, CDCl3): selected signals: 13.95 (C(O)OCH2CH3),
27.83 (C(CH3)3), 32.83 (CH2), 37.85 (CH2), 45.71 (CH2), 46.20 48.74 (CH), 49.04 (CH2), 50.38 (CH2), 51.48 (CH2), 52.59 (CH2),
(CH2), 46.45 (CH2), 52.11 (CH2), 52.20 (C(O)OCH3), 54.24 (CH), 52.95 (C(O)OCH3), 53.62 (NCH2CH), 53.62 (CH2), 54.86 (CH2),
58.17 (NCH2CH), 84.34 (C(CH3)3), 123.44 (Ar), 123.87 (Ar), 54.96 (CH2), 55.19 (CH2), 55.37 (CH2), 55.75 (CH2), 56.18
124.72 (Ar), 125.02 (Ar), 125.76 (Ar), 126.58 (Ar), 127.28 (Ar), (CH2), 60.62 (CH2), 60.94, 61.13, 61.21 (C(O)OCH2), 127.83
127.47 (Ar), 128.66 (Ar), 129.85 (Ar), 130.82 (Ar), 130.92 (Ar), (Ar), 128.12 (Ar), 128.41 (Ar), 131.53 (Ar), 131.84 (Ar), 132.62
131.33 (Ar), 151.86 (C(O), carbamate), 170.75 (C(O), ester). (Ar), 167.79 (C(O), amide), 170.29 (2 × C(O), ethyl ester), 170.52
HRMS (ESI): m/z: calcd for C35H45N4O5, [M + H]+, 601.3390, (C(O), methyl ester), 170.91 (C(O), ethyl ester). HRMS (ESI):
found: 601.3407.
m/z: calcd for
C
25H39N4O7, [M
+
H]+, 507.2813, found:
Synthesis of tris-alkylated nonanes 8, 9 and 10
Synthesis of bis(tert-butoxycarbonyl)amino-methoxycarbonyl-
507.2807.
Synthesis
of
1-(4-pyrenebutanamido)-carboxyethyl-4,7-bis-
ethyl-4,7-bis-(tert-butoxycarbonyl
methyl)-1,4,7-triazacyclo- (ethoxycarbonylmethyl)-1,4,7-triaza cyclononane – tris-alkylated
nonane – tris-alkylated nonane 8. tert-Butyl bromoacetate (85 μL, nonane 10. A solution of monoalkylated nonane 7 (0.69 g,
0.57 mmol) was added in one aliquot to a suspension contain- 1.16 mmol) in trifluoroacetic acid in dichloromethane (60%,
ing monoalkylated compound 5 (100 mg, 0.23 mmol) and 25 mL) was stirred overnight at room temperature. The solvent
K2CO3 (125 mg, 0.90 mmol) in MeCN (20 mL). The suspension was evaporated at reduced pressure. The residue was re-dis-
was vigorously stirred at room temperature for 3 hours. The solved in dichloromethane and the solvent was evaporated.
suspended solid was removed by filtration and the solvent was This procedure was repeated several times to give a light yellow
evaporated under reduced pressure. The residue was purified thick oil. 1H NMR spectroscopy (CDCl3) revealed the dis-
by flash chromatography (100% CH2Cl2 → CH2Cl2–EtOH appearance of the signals assigned to the Boc group in precursor
(50 : 50)) to afford tris-alkylated compound 8 (69 mg, 45%). 7. The oil (1.16 mmol, assuming quantitative deprotection)
8044 | Dalton Trans., 2014, 43, 8037–8047
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Dalton Transactions
Paper
was dissolved in MeCN (30 mL), K2CO3 (1.6 g, 11.5 mmol) was suspension was stirred gently at room temperature for 2 hours.
added and the suspension was vigorously stirred at room The wet resin was transferred into a chromatography column,
temperature for 15 minutes. To this suspension was added washed with water (∼50 mL) and eluted with hydrochloric acid
ethyl bromoacetate (0.31 mL, 2.77 mmol). The suspension was 0.1 M. The relevant fractions, identified by TLC (ethanol–water
vigorously stirred at room temperature overnight. The sus- 1/1, revelation with iodine vapor), were pooled, concentrated at
pended solid was removed by filtration, the solvent was evapor- room temperature and further dried under vacuum to afford
ated under reduce pressure and the residue was purified by ligand L2, in the hydrochloride form, as a light yellow solid
1
flash chromatography (100% CH2Cl2 → CH2Cl2–EtOH (1 : 1)) to (180 mg, 71%). H NMR (300 MHz, D2O): δ = 2.9–3.6 (broad,
1
afford compound 10 (0.425 g, 55%) as a white foam. H NMR overlapped signals with an integration corresponding to 14 H,
(300 MHz, CDCl3): δ = 1.26 (m, 6 H, 2 × C(O)OCH2CH3), 2.24 N(CH2)2N and NCCH2CH), 3.81 (broad, overlapped signals
(m, 2 H, NHC(O)CH2CH2), 2.44 (m, 2 H, NHC(O)CH2), with an integration corresponding to 4 H, NCH2CO2H), 4.80
2.60–2.90 (m, 4 H, NCH2C(O)), 2.50–3.40 (m, 16 H, N(CH2)2N, (m (broad), 1 H, CH), 7.53–7.88 (5 H, m, Ar). 13C NMR
NCH2C(O), NCH2CH), 3.37 (m, 2 H, NHC(O)(CH2)2CH2), 3.73 (75.4 MHz, D2O): selected signals: 43.02 (CH2), 48.11 (CH2),
(s, 3 H, C(O)OMe), 3.92 (m, 4 H), 4.10 (m, 6 H, C(O)OCH2), 48.93 (CH2), 49.03 (CH2), 49.78 (CH2), 51.39 (CH2), 51.71 (CH),
4.81(d(br), 1 H, NCH2CH), 7.80–8.40 (9 H, m, Ar). 13C NMR 51.91 (CH2), 54.53 (CH2), 56.08 (CH2), 56.77 (CH2), 127.46 (Ar),
(75.4 MHz, CDCl3): selected signals: 14.05 and 14.13 (C(O)- 129.04 (Ar), 132.60 (Ar), 133.32 (Ar), 170.20 (CO2H), 177.17
OCH2CH3), 27.23 (NHC(O)CH2CH2), 32.67 (NHC(O)(CH2)2- (C(O), amide). HRMS (ESI): m/z: calcd for C20H29N4O7,
CH2), 35.39 (NHC(O)CH2), 43.50 (CH2), 43.76 (CH2), 43.98 [M + H]+, 437.2031, found: 437.2027.
Synthesis of 1-(4-pyrenebutanamido)-carboxyethyl-4,7-bis-(carb-
oxymethyl)-1,4,7-triazacyclononane – fully deprotected NO2A-N-
(α-pyrenebutanamido)propionic acid ligand (L3). Prochelator (10)
(0.360 g, 0.53 mmol) was dissolved in a mixture of ethanol–
water (1/1 v/v, 20 mL). The solution was adjusted to pH ∼ 10 by
adding small portions of Dowex-1X2-100-OH− resin. The sus-
pension was kept under stirring at room temperature for
2 hours. The wet resin was transferred into a chromatography
column, washed with water (∼50 mL) and eluted with 0.1 M
hydrochloric acid, followed by a mixture of hydrochloric acid
0.2 M–ethanol (1/1 v/v). The relevant fractions, identified by
TLC (ethanol–water, 1/1 v/v, revelation with iodine vapor and
visualization under UV light λ365 nm), were pooled, concen-
trated at room temperature and further dried under vacuum to
afford deprotected chelator L3 in the hydrochloride form, as a
light yellow solid (0.149 g, 46%). 1H NMR (400 MHz, D2O/
CD3OD): δ = 1.9–3.3 (broad, overlapped signals with an
integration corresponding to 24 H, NHC(O)CH2CH2CH2,
NCH2CO2H, NCH2CH and N(CH2)2N), 4.49 (m (br), 1 H, CH),
7.80–8.40 (9 H, m, Ar). 13C NMR (75.4 MHz, D2O/CD3OD):
selected signals: 28.22 (NHC(O)CH2CH2), 33.25 ((NHC(O)-
(CH2)2CH2), 36.23 ((NHC(O)CH2), 48.92 (CH), 50.49 (CH2),
54.95 (CH2), 122.75 (Ar), 123.97 (Ar), 124.03 (CH-Ar), 124.58
(2 × (CH-Ar)), 125.71 (CH-Ar), 126.19 (CH-Ar), 126.93 (C-Ar),
126.99 (CH-Ar), 127.28 (CH-Ar), 127.92 (CH-Ar), 129.24 (C-Ar),
130.20 (C-Ar), 130.70 (C-Ar), 135.44 (C-Ar), 171.98 (CO2H),
172.19 (2 × CO2H), 175.41 (NHC(O)). HRMS (ESI): m/z: calcd
for C33H38N4NaO7, [M + Na]+, 625.2638, found: 625.2621.
(CH2), 48.06 (CH2), 51.22 (CH2), 51.72(CH2), 51.86 (C(O)OCH3),
52.51 (CH), 52.60 (CH2), 55.40 (CH2), 55.64 (CH2), 60.82, 60.98
(C(O)OCH2), 123.63 (Ar), 124.62 (Ar), 124.75 (Ar), 124.85 (Ar),
125.76 (Ar), 126.51 (Ar), 127.18 (Ar), 127.41 (Ar), 127.46 (Ar),
128.67 (Ar), 129.72 (Ar), 130.87 (Ar), 131.32 (2 × Ar), 136.43 (C),
136.53 (Ar), 171.36 (C(O), ethyl ester), 171.39 (C(O), ethyl ester),
171.54 (C(O), methyl ester), 173.64 (C(O), amide).
Synthesis of fully deprotected ligands L1, L2 and L3
Synthesis of aminocarboxyethyl-4,7-bis-(carboxymethyl)-1,4,7-
triazacyclononane – fully deprotected NO2A-N-(α-amino)propionic
acid ligand (L1). A solution of prochelator
8 (50 mg,
0.076 mmol) in a mixture of hydrochloric acid 6 M–ethanol
(12 mL; 2/1 (v/v)) was stirred at room temperature for 2 hours.
The solvent was evaporated under reduced pressure and the
residue was re-dissolved in a mixture of water–ethanol (10 mL;
1/1 (v/v)). The solution was adjusted to pH ∼ 10 with Dowex-
1X2100-OH− resin (10 mL, wet resin) and the suspension was
stirred for 1 hour at room temperature. The resin was trans-
ferred into a column, washed with water and further eluted
with hydrochloric acid 0.1 M. The relevant fractions, identified
by TLC (ethanol–water 1/1, revelation with iodine vapor) were
pooled together and evaporated at reduced pressure to afford
ligand L1 in the hydrochloride form as an off-white solid
(25 mg, quantitative yield).1H NMR (300 MHz, D2O): δ =
2.73–3.67 (m, 16 H, N(CH2)2N and NCH2CO2H), 3.06 (d, J =
3.9 Hz and x Hz, 1 H, ABX), 3.31 (dd, J = 12.3 and 3.9 Hz, 1 H,
ABX), 5.01 (dd, J = 10.5 and 3.9 Hz, 1 H, ABX). 13C NMR
(75.4 MHz, D2O): 48.71 (CH2), 49.04 (CH2), 49.50 (CH2), 50.93
(CH2), 51.68 (CH2), 53.35 (CH), 57.31 (CH2), 57.93 (CH2), 58.46
(CH2), 173.31 (CO2H), 174.99 (CO2H), 176.42 (CO2H). HRMS
(ESI) m/z: calcd for C13H25N4O6 [M + H]+: 333.1769, found:
333.1769.
Preparation of Ga3+ chelates for 1H and 71Ga NMR. To an
aqueous solution of the ligand (pH = 3.0) was added dropwise
an aqueous solution of GaCl3 in a 1 : 1 mole ratio. The pH was
kept below 3.8 by the addition of aqueous NaOH. The solution
was stirred at room temperature overnight and the solvent was
removed at reduced pressure. Solutions for NMR measure-
ments (20 mM, D2O) were obtained by dissolution of the solid
Synthesis of benzamido-carboxyethyl-4,7-bis-(carboxymethyl)-
1,4,7-triazacyclononane – fully deprotected NO2A-N-(α-benzoyl-
amido)propionic acid ligand (L2). Prochelator (9) (323 mg,
0.89 mmol) was dissolved in a mixture of ethanol–water
(20 mL, 1/1 (v/v)). The solution was adjusted to pH ∼ 10 by
adding small portions of Dowex-1X2-100-OH− resin. The
1
complexes in D2O (0.75 mL). H and 71Ga NMR spectra of the
complexes were obtained at 298 K on a Bruker Avance-3 400
Plus spectrometer, operating at 400.02 and 121.98 MHz,
This journal is © The Royal Society of Chemistry 2014
Dalton Trans., 2014, 43, 8037–8047 | 8045

Paper
Dalton Transactions
respectively. The proton NMR spectra of the complexes were
Groups of four animals (Wistar male rats weighting
assigned by 2D COSY and NOESY techniques using a Varian ca. 200 g) were anaesthetized with ketamine (50.0 mg mL−1)–
VNMRS 600 NMR spectrometer. The 1H and 71Ga NMR pH chloropromazine (2.5%) (10 : 3), injected in the tail vein with
study of the complexes was performed by adding small ca. 100 μCi of [67Ga]L3 and sacrificed 1 and 24 hours later. The
portions of NaOD or DCl to the complex solutions to obtain major organs were removed, weighed and counted in a γ well-
the desired pH.
counter. The biodistribution is stated as percentage of injected
dose per gram of organ (%ID g−1).
Fluorescence measurements
The absorption and the fluorescence emission spectra of the
free ligand L3 and the GaL3 complex were recorded, respecti-
vely, with a Jasco V-630 UV-Vis spectrophotometer and a
HORIBA Jobin Yvon Fluoromax-4 spectrofluorimeter, equipped
with a monochromator in both excitation and emission and a
temperature controlled cuvette holder. Fluorescence spectra
were corrected for the instrumental response of the system.
The fluorescence quantum yields (ΦF) were determined using
the standard method.52,53 Anthracene in ethanol (Φr = 0.27)54
was used as a reference.
Acknowledgements
This work was financially supported by Fundação para a
Ciência
e Tecnologia, Portugal (PEst-C/QUI/UI0686/2013;
FCOMP-01-0124-FEDER-037302; PTDC/QUI/70063/2006); grant
SFRH/BD/63994/2009 to Miguel Ferreira and sabbatical grant
SFRH/BSAB/1328/2013 to J. A. Martins; and Rede Nacional de
RMN (REDE/1517/RMN/2005) for the acquisition of the Varian
VNMRS 600 NMR spectrometer at the University of Coimbra
and the Bruker Avance-3 400 Plus at the University of Minho in
Braga. We also acknowledge the COST Action TD1004 “Therag-
nostics Imaging and Therapy”.
Radiochemistry
67Ga3+ complexes for in vivo and in vitro experiments were pre-
pared by adding 1 mCi of [67Ga]citrate (produced at IBA Mole-
cular, Louvain-la-Neuve) to a solution of 1 mg of the chelator
in HEPES buffer (400 μL, 0.4 M, pH 4) and heated at 80 °C for
ca. 1 hour. The radiochemical purity of the final products was
determined by TLC. The percentage of the bound metal was
above 95% in all cases.
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