1,4,7-Triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA): A new bifunctional chelator for radio gallium-labelling of biomolecules

Article abstract of DOI:10.1039/a801294f

A new bifunctional chelator NODASA (1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid) has been synthesised, its kinetically inert gallium(III) complex was crystallographically characterized and conjugated to a model aminoacidamide showing the fea

Full text of DOI:10.1039/a801294f

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1,4,7-Triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA): a new
bifunctional chelator for radio gallium-labelling of biomolecules
Joa˜o P. Andre´,a,b Helmut R. Maecke,*a Margareta Zehnder,c Ludwig Mackoc and Kayhan G. Akyeld
a Division of Radiological Chemistry, Institute of Nuclear Medicine, University Hospital, Petersgraben 4, 4031 Basel, Switzerland
b Department of Chemistry, University of Minho, Braga, Portugal
c Institute for Inorganic Chemistry, University of Basel, Basel, Switzerland
d Novartis Pharma AG, Basel, Switzerland
A new bifunctional chelator NODASA (1,4,7-triazacyclono-
nane-1-succinic acid-4,7-diacetic acid) has been synthesised,
its kinetically inert gallium(III) complex was crystallo-
graphically characterized and conjugated to a model ami-
noacidamide showing the feasibility of a prelabelling ap-
proach with 68,67Ga.
most dramatic effect in the 1H NMR spectrum of the chelate in
relation to the free ligand, which exhibits a singlet at d 3.50. The
sharp multiplets of the ethylenic protons might show the
existence of slow intramolecular processes of interconversion
CO2CHPh2
H
H
H
N
N
N
N
N
N
i
Over the past ten years a constant interest in the chemistry of
bifunctional chelators useful in biomedical applications has
been evident.1,2 Many were designed for coupling to mono-
clonal antibodies3,4 and other biomolecules.5 Recently bio-
active peptides were successfully introduced into the clinic for
in vivo visualization of tumours.6,7 As these peptides show very
fast blood clearance and diffusion into tissues the use of short
lived metallic positron emitters becomes possible. In this
context 68Ga (t1/2 = 68 min) is of special interest because its
half-life is compatible with the rate of localization of small
targeting molecules. For our purpose a bifunctional chelator is
needed which is comprised of a gallium immobilizing moiety
and a short carboxylate arm for coupling to the N-terminal end
of bioactive peptides. We aim at using this bifunctional chelator
in a preconjugation 67,68Ga-labelling approach of somatostatin
analogues. This preconjugation allows the covalent coupling of
a well defined radiometal complex of high specific activity; this
will be very important with regard to most radiopeptides
because of their potential pharmacological effects.
CO2CHPh2
N
H
N
H
ii
–
CO2CHPh2
CO2CHPh2
CO2
ButO2C
iii
N
N
–O2C
N
–
CO2
N
N
CO2But
–
CO2
iv
–
CO2
–O2C
N
–
CO2
v
Ga3+
N
21
22
24
15
CO2
–
20
23
3
2
10'
5
11
19
–
25
CO2
–O2C
14
N
N
12
16
C
N
H
NH2
Ga3+
17
18
9
N
O
Therefore we synthesised a new N-functionalised 1,4,7-tria-
zamacrocycle in three steps by alkylation of 1,4,7-triazacyclo-
nonane (Scheme 1): (i) with 1 equiv. of bis(diphenylmethyl)
d,l-bromosuccinate in chloroform; (ii) with 2 equiv. of tert-
butyl bromoacetate in acetonitrile in the presence of K2CO3
followed by deprotection with 6 m HCl (iii). The overall yield of
the synthesis of 1,4,7-triazacyclononane-1-succinic acid-4,7-di-
acetic acid (NODASA) was slightly > 50%.‡
6
8
O
10
–
CO2
13
Scheme 1 Synthesis of the chelate Ga(NODASA) and coupling to
d-phenylalanineamide
O(4)
C(1)
C(2)
Crystals of Ga(NODASA) were obtained from an aqueous
solution of the ligand and Ga(NO3)3 in equimolar amounts (pH
3, 70 °C, 1 h; Scheme 1 (iv), followed by slow evaporation.
Characterization by X-ray diffraction§ showed that the complex
is electrically neutral and the GaIII ion is fully chelated in a
slightly distorted octahedral environment (Fig. 1). The
b-carboxylate remains protonated and does not participate in the
complexation. The three nitrogen atoms of the triazacyclono-
nane define a plane in a facial arrangement and three of the
pendant carboxylate oxygens constitute another one. These
planes are almost coplanar with a dihedral angle of 1.75°. The
trans N–Ga–O bond angles average 165.4°. This leads to a
relative twist of the N3 and O3 planes by 14.6° away from a
symmetrically staggered conformation, similar to the parent
complex Ga(NOTA)8 and different previously synthesised
nickel(ii) and chromium(iii) NOTA complexes.9 The variation
in the individual Ga–O and Ga–N bond lengths is very small
showing overall high similarity to the parent complex Ga-
C(3)
C(12)
C(11)
N(1)
C(4)
O(2)
O(1)
Ga
C(6)
N(3)
O(5)
N(2)
O(7)
C(9)
C(8)
C(5)
C(7)
O(3)
C(10)
C(13)
C(14)
O(6)
O(8)
Fig. 1 ORTEP drawing of the Ga(NODASA) complex. Ga–N(1) 2.101(3),
Ga–N(2) 2.098(3), Ga–O(1) 1.937(3), Ga–O(3) 1.942(3) Å; O(1)–Ga–O(3)
94.7(1), O(2)–Ga–N(3) 165.6(1), O(2)–Ga–N(2) 82.5(1), N(2)–Ga–N(3)
84.1(1)°.
(NOTA).10 Ga(NODASA) was also characterized by its 1H, 13
C
and 69Ga NMR spectra.¶ A multiplet centered at d 3.75 (Fig. 2)
due to the magnetically non equivalent acetate hydrogens is the
Chem. Commun., 1998
1301

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–
In summary, 67,68Ga(NODASA) can be used in a pre-
labelling approach followed by conjugation to a biomolecule.
This approach is currently being followed using somatostatin
analogues.
This work was supported by the Swiss National Science
Foundation (No. 31-42516/94).
CO2
3
2
10′
11
8
10, 10′
5,6
3,8 (a)
–O2C
N
N
12
CO2H
Ga3+
N
3, 8 (b)
12
5
9
2, 9 (a)
6
2, 9 (b)
10
12′
–
CO2
11
Notes and References
† E-mail: maecke@ubaclu.unibas.ch
4.2
4.0
3.8
3.6
3.4
3.2
3.0
2.8
1
‡ The ligand had satisfactory elemental analysis, H, 13C NMR and mass
d
spectra. The pKa values were determined by pH potentiometry: pKHL
=
32
Fig. 2 1H NMR (400 MHz) spectrum of Ga(NODASA) in D2O, 7 mm, pD
= 3.6 and T = 22 °C
2
11.71, pKH L = 5.94, pKH L = 4.27, pKH L = 3.22 and pKH L = 1.95
22
+
2
3
4
5
(0.50 m KNO3).
§ Crystal data: C14H20GaN3O8·3H2O, monoclinic, space group P21/n [a =
7.6077(6), b = 20.573(3), c = 12.186(2) Å, b = 97.726(9)°], Z = 4, F(000)
= 1000, m = 2.56 mm21, Cu-Ka = 1.54180 Å, T = 293 K, qmax = 77.50°,
w–2q scan technique, 3561 independent reflections, 3011 used in
refinement, 284 parameters refined, final R = 5.08, final Rw = 0.0626,
Chebychev polynomial weighting. CCDC 182/850.
between the (lll) and (ddd) conformations11 of the ring
backbone arising from the high rigidity of the system.
Although the structural results give some important indica-
tions about the binding of GaIII to the macrocycle, the stability
of this chelate is very important for successful applications in
vivo. With a complexation competition method using 67Ga as a
radiotracer and NOTA∑ as an auxiliary competing ligand it has
been possible to estimate the conditional stability constant for
the complex at different pH values. The equilibration has been
followed for nine days. The determination of the ligand
protonation constants allows the calculation of the thermody-
¶
13C NMR 100 MHz (D2O), d 31.3 (C12), 44.9 (C2,9, 52.8–53.5
(C3,5,6,8), 61.9 and 62.0 (C10,10A), 65.8 (C11), 174.8 and 175.0
(C13,14,15,16). 69Ga NMR 72.05 MHz (D2O) shows a single resonance at
d + 165 (w1/2 = 1000 Hz).
∑ 1,4,7-Triazacyclononane-1,4,7-triacetic acid.
** O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate.
†† Rf[SiO2, isopropyl alcohol–NH3(aq) (7:3)]
=
0.40; m/z
namic stability constant of Ga(NODASA) to logKGa(NODASA)
=
(ESI+):574.4(MH+, 15), 594.1 (MNa+, 100); 13C NMR 100 MHz (D2O), d
32.0 (C12), 38.3 (C19), 44.0 (C2,9), 52.4–53.0 (C3,5,6,8) 54.4 and 54.8
(C18), 61.5 (C10,10A), 64.5 and 65.0 (C11), 128.4–128.9 (C21–25), 136.8
(C20), 169.4 (C17), 171.5 (C13,14), 172.4 (C15), 174.0 (C16).
30.9(0.2) compared to 30.98 of Ga(NOTA).12
An even more important indicator for in vivo applications is
the measurement of the rate of exchange of GaIII in blood serum
under the physiological conditions.14 For this experiment
67GaIII is first incubated with about 50 times excess of
NODASA at pH 6.2 in 0.5 m ammonium acetate buffer (25 min,
90 °C) in order to incorporate the metal ion. Then the complex
is mixed with blood serum and the exchange kinetics with
transferrin are measured at 37 °C. This was done by taking
aliquots of the serum, separating them by gel filtration, which
allows the separation of 67Ga(NODASA) from gallium(iii)–
transferrin (logK = 23.7),13 and measuring the activity in both
fractions with use of radiometric detection. The results clearly
show that 67Ga–NODASA virtually does not transfer any 67Ga
to transferrin over the observed period of 5 days, fulfilling the
criterion of high kinetic stability.
The kinetic stability of Ga(NODASA) with respect to the
acid-catalysed dissociation has been demonstrated with the aid
of the 67Ga complex, kept in 0.1 m glycine–HCl buffer, pH 2, at
37 °C. Aliquots of this solution were analysed by HPLC. After
5 days the complex was still 100% intact.
The fact that the b-carboxylate remains free while the other
three carboxylates are involved in five-membered chelate rings,
upon coordination to the metal ion, offers a very interesting
possibility to couple the chelate to a biomolecule. As a model
peptide we coupled d-phenylalanineamide to Ga(NODASA)
[Scheme 1 (v), in DMSO–DMF (2:1)] using HATU** 15 as the
coupling reagent with almost quantitative yield.†† HATU
allows coupling of carboxylate functions to primary amines
within minutes rendering even the coupling of 68Ga(NODASA)
to peptides feasible.
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Received in Basel, Switzerland, 16th February 1998; 8/01294F
1302
Chem. Commun., 1998