PIDAZTA: Structurally Constrained Chelators for the Efficient Formation of Stable Gallium-68 Complexes at Physiological pH

Article abstract of DOI:10.1002/chem.201901512

Two structurally constrained chelators based on a fused bicyclic scaffold, 4-amino-4-methylperhydro-pyrido[1,2-a][1,4]diazepin-N,N′,N′-triacetic acids [(4R*,10aS*)-PIDAZTA (L1) and (4R*,10aR*)-PIDAZTA (L2)], were designed for the preparation of Ga^III

Full text of DOI:10.1002/chem.201901512

A Journal of
Accepted Article
Title: PIDAZTA: Structurally Constrained Chelators for Efficient
Formation of Stable Gallium-68 Complexes at Physiological pH
Authors: Giovanni Battista Giovenzana, Edit Farkas, Adrienn Vágner,
Roberto Negri, Luciano Lattuada, Imre Tóth, Valentina
Colombo, David Esteban-Gómez, Carlos Platas-Iglesias,
Johannes Notni, and Zsolt Baranyai
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201901512
Link to VoR: http://dx.doi.org/10.1002/chem.201901512
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FULL PAPER
PIDAZTA: Structurally Constrained Chelators for Efficient
Formation of Stable Gallium-68 Complexes at Physiological pH
Edit Farkas,[a] Adrienn Vágner,[a] Roberto Negri,[b] Luciano Lattuada,[c] Imre Tóth,[a][d] Valentina
Colombo,[e] David Esteban-Gómez, Carlos Platas-Iglesias, Johannes Notni,* Zsolt Baranyai,*[a][c]
[f]
[f]
[g]
and Giovanni B. Giovenzana,*[b]
Dedicated to Prof. Silvio Aime in occasion of his 70th birthday
Abstract: Two structurally constrained chelators based on a fused
bicyclic scaffold, [(4R*,10aS*)-PIDAZTA (L1) and
4R*,10aR*)-PIDAZTA (L2)], were designed for the preparation of
The half-life of 68Ga matches the pharmacokinetics of low
molecular weight radiopharmaceuticals. Like other radiometal
7/68
III
(
ions, the introduction of 6 Ga in a diagnostic vector (peptides,
antibodies) does not rely on complex reaction sequences used
for lighter isotopes such as 18F or 11C, but rather requires a
III
Ga -based radiopharmaceuticals. The stereochemistry of the ligand
scaffold has a deep impact on the properties of the complexes, with
unexpected [Ga(L2)OH] species being superior in terms of both
thermodynamic stability and inertness. This peculiar behavior was
rationalized on the basis of molecular modeling and appears to be
[
4]
suitably designed chelating agent, which is covalently linked to
the vector[ and binds the radiometal by complex formation.
Ideally, such a chelator shows a high affinity and selectivity for
5]
III
III
related to a better fit in size of Ga into the cavity of L2. Fast and
Ga , along with rapid complexation kinetics matching the short
III
efficient formation of the Ga -chelates at room temperature was
lifetime of the radionuclide and high inertness to avoid
dissociation in the bio-fluids before excretion and/or radioactive
decay. However, many novel GaIII radiopharmaceuticals are still
prepared using the well-known chelating agent DOTA (1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid), despite its
68
observed at pH values between 7 and 8, which enables Ga
radiolabeling under truly physiological conditions (pH 7.4).
III
slow Ga complexation kinetics which is surpassed by many
Introduction
other chelators, for example, its hexadentate analogue NOTA
(
1,4,7-triazacyclononane-1,4,7-triacetic acid) and its bifunctional
There are two radioisotopes of gallium which are practically
[6a]
its phosphinate congener NOPO, [6b] HBED-CC,
derivatives,
[6c]
available for clinical application. While the γ-emitter 67Ga (t1/2
=
or polydentate siderophores.[6d-e] The main reason might be
7
8.3 h) only plays a subordinate role for scintigraphy, the clinical
the broad commercial availability of
functionalised DOTA derivatives for direct conjugation to specific
vectors with established protocols.[7]
a wide variety of
use of the positron emitter 68Ga (t1/2 = 67.7 min) has seen a
[
1]
particularly strong increase within the last decade. The broad
commercial availability of 68Ge/ Ga generators (small benchtop
devices providing 68GaIII in form of its hexaaqua complex in
dilute HCl)[2] and the high clinical value of some 68Ga-labelled
positron emission tomography (PET) radiopharmaceuticals,
above all, somatostatin analogs and prostate-specific membrane
antigen inhibitors, have firmly established 68Ga in clinical nuclear
medicine. [3]
68
[
[
[
a]
b]
c]
Dr. E. Farkas, Dr. A. Vagner, Prof. I. Toth, Dr. Zs. Baranyai, Dept. of
Inorganic and Analytical Chemistry University of Debrecen, H-4010,
Debrecen, Egyetem tér 1., Hungary
Dr. R. Negri, Prof. G.B. Giovenzana, Dip. di Scienze del Farmaco,
Università del Piemonte Orientale, Largo Donegani 2/3, 28100
Novara, Italy. Email: giovannibattista.giovenzana@uniupo.it
Dr. Zs. Baranyai, Dr. L. Lattuada, Bracco Imaging spa, Bracco
Research Centre, Via Ribes 5, 10010 Colleretto Giacosa (TO), Italy.
E-mail: Zsolt.Baranyai@bracco.com
Figure 1. AAZTA and derivatives studied for GaIII chelation.
In this context, the chelator AAZTA[8] (Fig. 1) was recently shown
to be a highly interesting scaffold, since it quickly forms
[d]
[e]
[f]
Prof. I Tóth, Dept. of Physical Chemistry University of Debrecen, H-
thermodynamically stable complexes with gallium(III)[9] and other
ions of diagnostic interest.[10] Bifunctional derivatives of AAZTA
4010, Debrecen, Egyetem tér 1., Hungary
Dr. V. Colombo, Dip. di Chimica, Università degli Studi di Milano,
Via Golgi 19, 20133 Milano, Italy
Dr. D. Esteban-Gómez, Prof. C. Platas-Iglesias, Centro de
Investigacións Científicas Avanzadas (CICA) and Dep. de Química,
Facultade de Ciencias, Universidade da Coruña, 15071 A Coruña,
Galicia, Spain
3
[11]
were used in conjugates with peptides (Tyr -Octreotide
and
Minigastrin[
12]
)
and
RGD-peptidomimetics
[13]
showing
6
8
III
accumulation in tumours of the corresponding Ga -chelates,
and with the bisphosphonate group for PET-imaging of bones.
Moreover, structural studies were devoted to shed light on the
coordination behaviour of the 1,4-diazepane ring and the
carboxymethyl side arms of AAZTA towards Ga . For this
[14]
[g]
Dr. J. Notni, Lehrstuhl für Pharmazeutische Radiochemie,
Technische Universität München, Walther-Meißner-Strasse 3, D-
85748 Garching, Germany
III
Supporting information for this article is given via a link at the end of
the document.
reason, the preparation of AAZTA derivatives with a lower
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Chemistry - A European Journal
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FULL PAPER
denticity, termed DATAs[15] (Fig. 1) and different coordinating
arms[16] was reported to better understand the role of donor
groups in the coordination of the classically hexacoordinated
The identity of the isomers was assessed by single crystal X-ray
diffraction analysis of nitrodiamine 3, thus establishing its
(4R*,10aS*) stereochemistry (Fig. S1, S2), and assigning the
configuration (4R*,10aR*) to the diastereomeric pair 4. Parallel
III
Ga . The results of the study of their coordination properties
highlighted the role of the side arms (number and steric
hindrance), and even more importantly, the ring substitution
pattern, on the overall stability of the corresponding GaIII
2
treatment of the separated isomers with H on Pd/C resulted in
the combined hydrogenation-debenzylation leading to the
triamines 5 and 6. The introduction of the carboxymethyl side
arms was achieved by an exhaustive alkylation with t-butyl
chelates.[17,18] For example, the change from R = CH
to R = Ph
3
in the structure of DATA affects the conformational population of
2 3
bromoacetate in acetonitrile in the presence of K CO ,
the seven-membered ring and accordingly the affinity for the
followed by the removal of the t-butyl groups with neat TFA to
obtain the desired chelating agents L1 and L2, respectively.
metal ion.[
19,20]
A careful design of the structure of the ligand is
therefore crucial in the search for improved and more efficient
chelating agents for GaIII.
The strong affinity of AAZTA and its derivatives for GaIII
combined with the fast kinetics of the complex formation
prompted us to explore the possibility to improve these
promising properties by designing new related chelating agents.
For this purpose, we have chosen to pursuit the conformational
locking of the 6-amino-1,4-diazepane substructure of this family
of chelating agents by ring-fusion with an additional six-
membered ring. The ring-fusion was planned to include one of
the coordinating nitrogen atoms, to maximise its influence on the
restricted conformational freedom of the complex arising from
the hexadentate chelating agent. A similar strategy was recently
reported for
a conformationally locked AAZTA derivative
(
CyAAZTA), leading to a heptadentate ligand in which the ring
fusion does not directly involve coordinating atoms in any of the
key bridgehead positions.[21]
In the present work, we report on the preparation of two novel
isomeric chelating agents, i.e.: 4-amino-4-methylperhydro-
pyrido[1,2-a][1,4]diazepin-N,N',N'-triacetic acids, (“PIDAZTAs”,
isomers hereinafter referred to as L1 and L2) depicted in
Scheme 1, along with the synthetic route adopted for their
preparation. We investigated thermodynamics, transmetallation
III
II
II
II
kinetics and structural properties of the Ga -, Ca -, Mn -, Zn -,
II
III
Cu - and Ln - complexes formed with the two PIDAZTA isomers.
Furthermore, in order to elucidate their practical value for
application in Ga radiopharmaceuticals, both isomers were
characterized in terms of 68Ga labelling properties and kinetic
inertness of the resulting radiometal complexes.
68
Results and Discussion
The synthesis of the chelating agents (Scheme 1) starts from
commercially available (±)-2-(aminomethyl)piperidine (1), whose
primary amino group was selectively N-benzylated through
sequential formation of the imine by dehydrative condensation
with benzaldehyde in dichloromethane, followed by reduction
Scheme 1. Synthetic preparation of the chelating agents L1 and L2.
Diffractometric analysis of a single crystal of L1 (Fig. 2, S3, S4),
grown by evaporation of a solution in methanol, confirmed the
relative stereochemistry of this isomer as (4R*,10aS*) and its
expected retention through the entire synthetic process. The
compound is chiral and crystallizes in the centric space group
4
with NaBH to diamine 2. The latter was subjected to a double
nitro-Mannich reaction with (para)formaldehyde and nitroethane,
leading to the formation of the isomeric bicyclic nitrodiamines 3
and 4, easily separated by column chromatography. The
availability of both isomers is crucial for a comprehensive
evaluation of the ring fusion influence on the resulting
P2
additional details are reported in the ESI). Fig. 2 shows the
absolute configuration of the stereocentres for the arbitrary
1
/c as a racemate, with one molecule per asymmetric unit
(
III
complexing ability towards Ga .
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choice of one enantiomer. In this structure, the piperidine ring is
found to be in a chair conformation, whereas the seven-
membered ring has a chair-like conformation. The pendant
carboxylic groups are employed to set up strong H-bond
contacts. Indeed, in the solid state, one intra-molecular H-bond
of the type O-H O (2.531 Å) is found between two pendant,
carboxymethyl groups of the exocyclic iminodiacetic (IMD)
residue, whereas the third carboxymethyl arm, located on the
endocyclic nitrogen atom, is involved in an inter-molecular HB
Table 1. Stability and protonation constants (logK) of [Ga(L1/L2)],
m
[Ga(DATA )], [Ga(CyAAZTA)], [Ga(AAZTA)] and [Ga(NOTA)]
complexes (0.15 M NaCl, 25C).
.
..
GaL
Ga(HL)
Ga(H
2
L)
Ga(L)OH
log
Ga(L)OH
18.77(3)
2.41(3)
−
4.04 (4)
14.74 (4)
[
[
Ga(L1)]
Ga(L2)]
21.70(4)
21.54
2.51(3)
2.42
4.09
3.14
0.9
−
−
3.75 (3)
6.25
17.94 (3)
15.29
(
2.603 Å) connecting the molecules in collinear chains, running
m
[a]
[
Ga(DATA )]
in the [001] crystallographic direction (Fig. S4). L1 is in its
zwitterionic form, in which the deprotonation of one carboxylic
group is observed, with concomitant protonation of the nitrogen
atom involved in the ring fusion.
[
Ga(CyAAZTA)]-[b]
Ga(AAZTA)]-[c]
Ga(NOTA)][d]
21.39
2.32
1.14
−
7.31
14.08
[
21.15
4.60
16.57
[
29.60
9.83
19.77
[
a] Ref. [17]. [b] Ref. [21]. [c] Ref. [9]. [d] Ref. [26] (0.1M TMACl, 298K).
III
The stability constants of Ga -complex formed with L2 is higher
by 3 logK units than that of L1, which might be explained by the
H
higher total basicity of L2 (logK
i
, Table S3) and their different
structural properties. The logKGaL values of Ga(L2) is similar to
m
-
-
those of [Ga(DATA )], [Ga(CyAAZTA)] and [Ga(AAZTA)] . In
III
III
Ga -complexes formed with L1/L2, the Ga ion is coordinated
by three N and three O donor atoms. However, the structure of
the pre-organised coordination cage formed by the donor atoms
in L1/L2 is clearly influenced by the rigidity of the fused
heterobicyclic skeleton. By considering the logKGaL values of
L1/L2, it can be assumed that the coordination cage of L2
provides a significantly favourable coordination environment for
Figure 2. X-Ray structure of (4R*,10aS*)-PIDAZTA (L1). Thermal ellipsoids of
non-H atoms were drawn at the 50% probability level. Intra-molecular H-bonds
are depicted with dashed lines. Colour codes: O, red; N, blue; C, gray; H,
white.
III
the Ga -ion in the corresponding complex. Interestingly, the
-
-
formation of [Ga(L1)OH] and [Ga(L2)OH] species (logKGa(L)OH
,
Table 1) occurs at notably lower pH values than that of
2
-
5m
-
2-
[
[
[
Ga(AAZTA)OH] , [Ga(DATA )OH] , [Ga(CyAAZTA)OH] and
Ga(NOTA)]. A comparison of the logGa(L)OH values of the
Ga(L)OH] species predominant at physiological conditions
3
+
Solution equilibrium properties of the Ga -L1/L2 systems
shows that [Ga(L2)OH]- is characterized by the highest
cumulative stability constant among the Ga -complexes formed
The in vivo application of 68Ga isotope requires very robust
III
6
8
III
Ga -complexes, which must be characterised by high
with AAZTA derivatives. The equilibrium data obtained by the
pH-potentiometric titration have been used to calculate the
thermodynamic stability and kinetic inertness in order to limit the
transmetallation or transchelation reactions with competing
endogenous species.[4,22-25] The stability and protonation
constants of the [Ga(L1/L2)] complexes have been calculated by
using the data obtained with pH-potentiometry, 1H- and 71Ga-
III
III
species distribution diagram for the Ga -L1 and Ga -L2
III
systems (Fig. 3 and 4). Amount of Ga aq, [Ga(OH)
4
] and
1
71
Ga(L)OH species achieved by H- and Ga-NMR studies of the
III
III
Ga -L1 and Ga -L2 systems are also shown in Fig. 3 and 4.
The species distribution diagrams, the H- and 71Ga-NMR
spectra (Fig. 3, 4, S7-S10) indicate that the complex formation is
complete for [Ga(L1)] and [Ga(L2)] at pH2.0 and pH1.5,
respectively. The Ga-NMR signal of the highly symmetric
species is relatively sharp at -log[H ]= 0.0 in the
Ga -L1 and Ga -L2 systems (Fig. S7 and S9, 
intensity of the Ga-NMR signals decreases by increasing the
pH due to the formation of protonated [Ga(HL1)]+ and
Ga(HL2)] species in the -log[H ] ranges 1-2 and 0.5-1.5,
1
71
III
NMR spectroscopy. H- and Ga-NMR spectra of the Ga -L1
1
III
and Ga -L2 systems at different pH values are reported in Fig.
S7-S10. The stepwise protonation constants of the free L1/L2
ligands (logKH
) used for the calculation of the stability and the
i
7
1
protonation constant of [Ga(L1/L2)] complexes are reported in
Table S3. The stability and protonation constants of [Ga(L1/L2)]
complexes obtained by pH-potentiometry, 1H- and Ga-NMR
spectroscopy are listed and compared with those of
3
+
+
[
Ga(H
III
2
O)
6
]
III
½
= 55 Hz). The
71
7
1
m
-
-
[
Ga(DATA )], [Ga(CyAAZTA)] , [Ga(AAZTA)] and [Ga(NOTA)]
+
+
[
in Table 1. (The experimental details, the definitions and
equations used for the evaluation of the equilibrium data and the
respectively. In the pH range of 2-3, the deprotonation of the
+
1
[
Ga(HL)] species results in a slight upfield shift of all H-NMR
II
II
II
II
III
equilibrium characterization of Ca -, Zn -, Mn -, Cu - and Ln -
signals which might be explained by the fact that the protonation
of [Ga(L1/L2)] complexes takes place at the weakly coordinated
carboxylate group of the ligand.
complexes formed with L1 and L2 are summarized in ESI).
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Chemistry - A European Journal
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FULL PAPER
coordinated by three N, two exocyclic carboxylate O donor
-
atoms and the OH ion (the acetate pendant arm located on the
endocyclic nitrogen atom does not participate in the coordination
of the GaIII ion, see DFT calculations below). At pH>8.5, the
-
III
competition of L1/L2 with OH ion for Ga takes place by the
appearance of the 71Ga-NMR signal of the [Ga(OH)
]- (Ga=223
4
1
ppm, 
of the free L1/L2 ligands.
The intensity of the 71Ga-NMR signal of the [Ga(OH-)
increases by increasing pH due to the dissociation of
½
= 90 Hz, Fig. S8 and S10) and by the H-NMR signals
]- species
4
-
-
[
Ga(L1)OH] and [Ga(L2)OH] species in the pH ranges 7.5-10.5
and 9.0-11.5, respectively. In the Ga -L1 system, the Ga(OH)
III
3
Figure 3. The percentage of the Ga3+aq (◆), [Ga(L)OH] () and [Ga(OH)
(
system. Chemical shift of the -CH protons () as a function of pH. The
3
species distribution was calculated from the equilibrium data (Tables 1 and
S3) obtained by pH-potentiometric titration ([Ga3+] = [L1] = 10.0 mM, 0.15 M
NaCl, 25C).
-
]-
species is formed in relatively large quantity due to the low
4
) vs. pH calculated from the 1H- and 71Ga-NMR spectra of the Ga -L1
III
-
1
stability of the [Ga(L1)OH] species. The H-NMR signal of the
CH - protons of free and complexed L1/L2 are well separated
3
with chemical shifts of 0.8 and 1.0 ppm, respectively. The
deprotonation of the free L1/L2 ligands results in a significant
upfield shift of CH
protons in [Ga(L1)OH]- and [Ga(L2)OH]- species remains
unchanged. The intensity of the CH - protons of the Ga(L1/L2)
3 3
- protons, whereas the frequency of CH
3
complexes decreases with the increase of pH due to the
-
dissociation of the [Ga(L1/L2)OH] species and the formation of
-
the [Ga(OH)
4
] (Fig. S8 and S10). The stability and protonation
constant of Ga(L1) and Ga(L2) complexes obtained by the pH-
potentiometry and multinuclear NMR spectroscopy) are in very
good agreement (Table S5).
Dissociation kinetics in solution
To evaluate the potential application of [Ga(L1/L2)] complexes
as 68Ga based radiodiagnostics for PET imaging, their kinetic
inertness must be assessed, because the high kinetic inertness
of the 68Ga complexes is especially important for targeting
purposes to guarantee the delivery of the radioisotope in the
Figure 4. The percentage of the Ga3+aq (◆), [Ga(L)OH] () and [Ga(OH)
(
-
]-
4
) vs. pH calculated from the H- and 71Ga-NMR spectra of the Ga -L2
system. Chemical shift of the –CH protons () as a function of pH. The
species distribution was calculated from the equilibrium data (Tables 1 and
S3) obtained by pH-potentiometric titration ([Ga3+] = [L2] = 10.0 mM, 0.15 M
NaCl, 25C).
1
III
3
III
form of intact complex to the target organ or tissue. The Ga -
complexes are generally characterized by high thermodynamic
stability. Then, their kinetic properties are often measured in
Generally the line-width of 71Ga-NMR signals ( Ga is
71
quadrupolar nucleus) is strongly influenced by the symmetry of
[
26-
strong acidic ([H+]>1.0 M) and basic conditions ([OH-]>0.1 M).
III
m
Ga -complexes ([Ga(DATA )]: Ga=129 ppm, 
½
= 1000 Hz;
= 4700 Hz, 308 K and
2
8]
-
[
Ga(CyAAZTA)] : Ga=119 ppm, 
Ga(AAZTA)] : Ga=118 ppm, 
½
III
Generally, the base catalysed dissociation of Ga -complex is
significantly faster than that of acid catalysed processes due to
-
[9,17,21]
[
½
= 2200 Hz).
Moreover, the
broadening or sharpening of the 71Ga-NMR signal could be
interpreted by the interaction of the nuclear quadrupole moment
with the electric-field gradient at the 71Ga nucleus.[27] At pH=3.5,
the 71Ga-NMR signals of [Ga(L1/L2)] complexes are very broad
and not observable, as a result of the asymmetric coordination
environment of the Ga3+ ion provided by the rigid structure of
III
[26-28]
the hydrolytic properties of Ga -ion.
However, the
conditions of such studies differ considerably from the
physiological ones and the use of the results obtained in these
experiments to predict the behaviour of GaIII complexes in body
fluids can be quite risky. Body fluids are very complex media, i.e.
endogenous metal ions and ligands may exchange with the
L1/L2 (Fig. S8 and S10). At pH>3.0 the formation of the
Ga(L)OH] species is evidenced by the extra equivalent base
68
components of the administered
Ga complexes in
-
[
transmetallation or transchelation reactions. The possibility of
consumption during the pH-potentiometric titration and the shift
6
8Ga releasing after in vivo administration of the complex, is
of all the signals to lower frequency in the 1H-NMR spectrum
determined by thermodynamic relations, expressed by the
stability constants of the different complexes formed in body
fluids. Based on the equilibrium data (Tables 1, S3, S4), the
-
(
Fig. S8 and S10). In the pH range 6-8, the [Ga(L)OH] species
III
III
predominates in both Ga -L1 and Ga -L2 systems. Since the
1
-
-
H-NMR spectra of the [Ga(L1)OH] and [Ga(L2)OH] contain
II
II
endogenous metal ions (mainly Cu and Zn ) or serum proteins
such as transferrin may compete with [Ga(L1/L2)] complexes
resulting in the dissociation of the latter. On these premises, the
several broad multiplets, it can be assumed that the [Ga(L)OH]-
species of both GaIII complexes are quite rigid structures. In the
-
-
III
[
Ga(L1)OH] and [Ga(L2)OH] species the Ga ion is presumably
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Chemistry - A European Journal
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FULL PAPER
transmetallation and transchelation reactions with CuII and
transferrin have been studied by spectrophotometry close to
physiological conditions for the [Ga(L1/L2)] complexes.
spontaneous dissociation of [Ga(L1/L2)OH] , [Ga(DATA )OH] ,
-
m
-
[Ga(CyAAZTA)OH]2- and [Ga(AAZTA)OH]2- presumably occurs
by the intramolecular rearrangement of the Ga -complexes with
III
Transmetallation reactions occurring between [Ga(L1/L2)]
complexes and CuII ions have been investigated by
the stepwise de-coordination of each donor atom and
III
consequent release of the Ga -ion. The very slow spontaneous
spectrophotometry at high [Ga(L1/L2)] excess ([GaL]tot/[CuII]tot
=
dissociation of [Ga(L2)OH] can be ascribed to its configuration
-
1
0 and 20) in the presence of citrate to prevent the hydrolysis of
providing a favourable and rigid coordination environment for the
III
II
III
the released Ga and the exchanging Cu ions in the pH range
.0-9.0. The k pseudo-first-order rate constants characterizing
Ga -ion, leading to slower intramolecular rearrangements and
6
d
OH- assisted dissociation processes. Using the rate and
equilibrium constants presented in Table 2, the half-lives
the transmetallation reactions of Ga(L1/L2) complexes (Fig.
S13) indicate that the rate of the transmetallation reaction is
directly proportional to the OH- concentration but does not
(t1/2=ln2/k
d
) of the dissociation reactions of [Ga(L1)OH]- and
-
[Ga(L2)OH] at pH=7.4 have been calculated and compared with
II
m
-
[Ga(CyAAZTA)OH]2-
depend on [Cu ]. Accordingly, the reactions occur through the
that
of
[Ga(DATA )OH] ,
and
-
[Ga(AAZTA)OH] complexes. The t1/2 values of [Ga(L1)OH]-,
2-
spontaneous (k
0
) and the OH -ion assisted (kOH) dissociation of
the dominant [Ga(L1))OH] and [Ga(L2)OH] species (Scheme 2), [Ga(L2)OH] , [Ga(DATA )OH] , [Ga(CyAAZTA)OH]2- and
-
-
-
m
-
followed by a fast reaction between the released L1/L2 ligand
and the free Cu ions. However, the k
[Ga(AAZTA)OH]2- are 0.27, 295, 11.2, 8.5 and 21 hours,
II
-
-
d
vs. [OH ] curve for
respectively, highlighting that [Ga(L2)OH] is characterised by
-
[
Ga(L1)OH] is a saturation curve (Fig. S13), which can be
the highest kinetic inertness, due to a very slow spontaneous
-
interpreted by assuming the formation of the di-hydroxo
Ga(L1)(OH)
]2- intermediate characterized by the KGa(L)(OH)2
equilibrium constants. Unfortunately, the results of the
and OH -assisted dissociation. The dissociation of the “gold
[
2
standard” [Ga(NOTA)] is significantly slower (several weeks at
pH>13)[27] than that of [Ga(L2)OH] . However, [Ga(L2)OH] is
characterized by the highest kinetic inertness among the GaIII-
complexes formed with AAZTA-like ligands.
-
-
1
71
equilibrium studies (pH-potentiometry, H and Ga-NMR) could
not confirm the formation of the [Ga(L1)(OH)
]2- intermediate,
2
which might be characterized by a very low kinetic inertness due
to the coordination of only 4 donor atoms of the ligand to the
GaIII ion. By taking into account these considerations, the
Table 2. Rate (k) and equilibrium (K) constants and half-life values (t1/2=ln2/k
d
)
2
-
characterizing the transmetallation and the transchelation reactions of
spontaneous dissociation of the [Ga(L1)(OH)
more probable, characterized by the kOH rate constants. The
2
]
intermediate is
m
-
-
II
2
[Ga(L1/L2)], [Ga(DATA )], [Ga(CyAAZTA)] and [Ga(AAZTA)] with Cu and
transferrin (25C)
mechanisms of the transmetallation reactions for the
-
-
Ga(L1)
Ga(L2) Ga(DATA )
m
[a] Ga(CyAAZTA)[b] Ga(AAZTA)[c]
[
Ga(L1)OH] and [Ga(L2)OH] are summarized in Scheme 2.
1
.40.1
4.3 0.2
−1
k0 /s
8.0∙10-6
1.7∙10-5
3.0∙10-6
-
4
-7
∙
10
∙10
−
1
−1
k
OH / M s
−
0.60.1
31
68
10
−
1
−1
1.8 0.2
kOH2 /M
s
−
−
−
−
−
−
−
-
3
∙
10
1
.5
K
Ga(L)(OH)2
/
−
6
-
1
0.3∙10
M
-
1
k
d
/ s
(7  1)
(6.50.3)
-
5
-5
-6
1
.7∙10
2.3∙10
9.2∙10
-
4
-7
(
pH=7.4)
∙10
.270.05
50 50
∙10
t
1/2 / h
0
29515
11.2
8.5
21
Scheme 2. Proposed mechanism of the transmetallation reactions for the
(pH=7.4)
[
[
Ga(L1)] and [Ga(L2)] complexes. The reaction path in the middle is valid for
Ga(L1)] complex only.
6
d
k  10
6

0.7

0.1
21
29
8
-
1
s
[
d]
d]
(
(
sTf exch.)
t
1/2 / h
The rate and stability constants characterising the
0.29
283
9.4
6.6
24
[
sTf exch.)
II
transmetallation reaction of [Ga(L1)] and [Ga(L2)] with Cu are
m
[a] Ref. [17]. [b] Ref. [21] (0.1M KCl). [c] Ref. [9] (0.1M KCl). [d] 0.025M NaHCO
M NaCl, 25°C, pH = 7.4.
listed
[
and
compared
with
those
of
[Ga(DATA )],
3
,
Ga(CyAAZTA)] and [Ga(AAZTA)]- complexes in Table
-
2
(
Experimental details, definitions and equations used for the
evaluation of the kinetic data are summarized in ESI).
The rate constants summarized in Table 2 indicate that
Transchelation reactions with human serum transferrin: It is well
established that GaIII is transported through the circulatory
-
spontaneous (k
[
[
0
)
and OH -assisted (kOH
)
dissociation of
system by the serum iron transport protein transferrin due the
-
-
Ga(L2)OH] is significantly slower than that of [Ga(L1)OH] and
Ga(CyAAZTA)OH]2- complexes. Interestingly, the k
rate constants of [Ga(L2)OH] are one order of magnitude lower
[
29,30]
relatively high concentration of transferrin in human plasma
0
and kOH
III
III
and the strong affinity of Ga to transferrin (Ga -transferrin:
logKGaTf=18.9, logKGa2Tf=17.7).[31] Since human serum transferrin
-
2
-
than the corresponding rate constant of [Ga(AAZTA)OH] . The
III [32]
is normally 30% saturated with Fe ,
it has a relatively high
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Chemistry - A European Journal
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FULL PAPER
capacity to bind GaIII and to compete with ligands for the GaIII-
ion, thus resulting in the in vivo dissociation of the dominant
(Fig. 5, see also Table S6). These results suggest that the
presence of the additional six-membered ring in the bicyclic
scaffold of the ligand introduces some steric hindrance for the
coordination of N1, causing a concomitant lengthening of the
trans Ga-O2 distance. This effect is more pronounced in
[Ga(L1)] than in [Ga(L2)], which explains the lower stability of
the former. This is in line with the relative free energies obtained
with DFT, which predict a higher stability of [Ga(L2)] by 7.5 kJ
-
-
[
Ga(L1)OH] and [Ga(L2)OH] species. To assess the extent of
III
-
Ga release in the competition reactions of [Ga(L1)OH] and
Ga(L2)OH]- with transferrin, the ligand exchange reactions
between Ga -complexes and human serum transferrin (sTr)
have been studied by spectrophotometry at the absorption band
of the Ga -sTf complex in the 240-250 nm range. The rate
[
III
III
mol . A more detailed analysis of the GaIII coordination
environment was carried out by calculating the electron density
() at the bond critical points (BCPs), which can be correlated to
-1
d d
constant (k ) and half-life (t1/2=ln2/k ) values characterizing the
-
-
trans-chelation reactions of [Ga(L1)OH] , [Ga(L2)OH] and sTf
m
are shown and compared with those of [Ga(DATA )],
Ga(CyAAZTA)]-
and
Experimental details, definitions and equations used for the
evaluation of the kinetic data are summarized in ESI).
The k rate constants obtained for the ligand exchange reaction
of [Ga(L1)OH]-, [Ga(L2)OH]- with sTf were found to be
[Ga(AAZTA)OH]2-
in
Table
2.
the strength of the metal-donor bonds. The results (Fig. 5 and
[33]
[
(
Table S7) show that the Ga-O bonds are stronger than the Ga-N
ones, as would be expected. The value of BCP calculated for the
Gd-N1 bonds is lower for the complex of L1, confirming a
weaker binding of N1 in [Ga(L1)] than in [Ga(L2)].
The 71Ga NMR chemical shifts of [Ga(L1)] and [Ga(L2)]
were calculated using relativistic DFT calculations (see
computational details below) following a methodology similar to
that employed recently to predict 89Y NMR shifts.[34] To assess
the accuracy of our calculations, we also computed the chemical
d
(
65050)10-6 s-1 and (0.70.1) 10 s , which are essentially
-6
-1
equal to those obtained in the metal exchange reaction between
-
-
II
[
Ga(L1)OH] , [Ga(L2)OH] and Cu in the presence of citrate
-
=72010-6 s ; [Ga(L2)OH] : k =0.6510-6
-1
-
excess ([Ga(L1)OH] : k
d
d
-1
s , 0.15 M NaCl, 25C). These findings can be interpreted by
assuming that sTf has practically no effect on the rate of the
trans-chelation reactions, which has been controlled by the
spontaneous and hydroxide assisted dissociation of [Ga(L1)OH]-
and [Ga(L2)OH]- complexes followed by the fast reaction
between the released Ga -ion and sTf. The comparison of the
) calculated for the Ga -complexes indicates that the
kinetic inertness of [Ga(L2)OH] is about 1000, 30, 43 and 13
m
-
shifts of [Ga(DATA )], [Ga(NOTA)] and [Ga(OH)
4
] . For chemical
71
shift calculation purposes we computed the isotropic Ga
nuclear shielding of [Ga(H O)
2 6
]3+, so that the systems chosen for
Ga NMR chemical shifts calculations cover a chemical shift
range of 223 ppm (Table 3).
71
III
III
half-life (t
½
-
-
m
times higher than those of the [Ga(L1)OH] , [Ga(DATA )],
Ga(CyAAZTA)] and [Ga(AAZTA)OH]2- complexes, respectively.
-
Table 3. Experimental 71Ga NMR chemical shifts (exp) and linewidths (
[
½
) ,
71
71
isotropic Ga nuclear shielding values (iso
) and calculated Ga NMR
calc
chemical shifts ( ) obtained with relativistic DFT calculations.
DFT study
exp (ppm)
calc (ppm)
Complex

½
(Hz)

iso (ppm)
1901.8
1782.5
1779.3
1778.8
1741.1
1653.0
[
[
Ga(H
2
O)
6
]3+
0
150
0
Aiming to rationalise the different stability constants and
dissociation kinetics of the Ga complexes with L1 and L2, we
performed DFT calculations at the TPSSh/TZVP level (see
computational details below). These calculations provided
optimized geometries containing six-coordinated Ga ions,
which are directly bound to the three nitrogen atoms of the
ligand and three oxygen atoms of the carboxylate groups. The
metal coordination environments in both L1 and L2 can be
described as distorted octahedral. The triangular face defined by
the three coordinated oxygen atoms defines an angle of 9.9°
III
a
a
Ga(L1)]
119
122
123
161
249
[Ga(L2)]
119
129
170
223
20000
1000
210
III
m
b
[
[
[
Ga(DATA )]
Ga(NOTA)]c
Ga(OH)
4
]-
90
[
a] Too broad to be observed. [b] Ref. [20]. [c] Ref. [27]
.
(
L1) and 8.7° (L2) with the plane delineated by the three
nitrogen atoms of the ligand. The mean twist angles of these two
triangular faces (49.4+8.6° and 50.6+8.0° for L1 and L2,
respectively), reflect a certain degree of distortion from an
octahedron (ideal value 60°) towards a trigonal prism (ideal
value 0°). Analogous calculations performed for [Ga(NOTA)]
evidence a more symmetrical coordination environment, with
Initial test calculations indicated that the agreement between
71
experimental and calculated
Ga NMR shifts improved
considerably when explicit second sphere water molecules were
]- and [Ga(H ]3+. Thus, we
included in the models of [Ga(OH)
performed calculations using
4
O)
2 6
a
mixed cluster-continuum
virtually parallel O
The Ga-N and Ga-O distances calculated for [Ga(NOTA)] are
.135 and 1.943 Å, respectively. The Ga-N and Ga-O distances
3
and N
3
planes and a twist angle of 46.7+0.0°.
]-·8H
3+
approach on the [Ga(OH)
4
2
O and [Ga(H
2
O)
6
2
] ·12H O
systems.[35]
2
involving N2, N3, O1 and O2 in the L1 and L2 complexes are
m
similar to those of obtained for [Ga(NOTA)] and [Ga(DATA )],
while the Ga-N1 and Ga-O2 distances are considerably longer
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Chemistry - A European Journal
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FULL PAPER
which is directly linked to kinetic inertness, and the experimental
conditions required for radiometal complexation (labelling).
Regarding
the
latter,
the
necessity
to
produce
radiopharmaceuticals with sufficiently high molar activity[
requires efficient radiometal incorporation at the lowest possible
chelator concentration. Furthermore, rapid complex formation at
ambient temperature and physiological pH is an attractive
feature, being indispensable for labelling of targeting vectors that
do not tolerate elevated temperatures or challenging pH values,
e.g. large proteins like antibodies, which is however of lesser
36]
68
relevance for the short-lived Ga. More importantly, such
properties would provide access to 68Ga tracers via “shake-and-
shoot” kits, that is, by simple addition of 68Ge/ Ga generator
eluate to pre-conditioned vials containing lyophilized precursor
68
and excipients, as known from 9 Tc radiopharmaceuticals.
9m
[37]
Figure 5. Geometries of the [Ga(L1)] (a) and [Ga(L2)] (b) complexes
optimized at the TPSSh/TZVP level. Bond distances of the metal coordination
environments and electron densities at the corresponding bond critical points
are also provided.
Our DFT calculations provide 71Ga NMR chemical shifts in good
agreement with the experimental values, the largest deviation
being observed for the [Ga(OH)
4
]- complex. The chemical shift
calculated for [Ga(L2)] (122 ppm) is in excellent agreement with
the experimental value (119 ppm), which supports that our DFT
calculations provide a good model of the structure of this
complex in solution. The chemical shifts calculated for [Ga(L1)]
and [Ga(L2)] are very similar, as would be expected given the
very similar coordination environment of GaIII in the two
complexes. Geometry optimizations of the hydroxo complexes
reveals that one of the carboxylate groups of the ligand remains
uncoordinated, with a hydroxide anion completing the six-
coordination environment around the metal ion. The
-
-
[
Ga(L1)(OH)] and [Ga(L2)(OH)] complexes present very similar
bond distances of the metal coordination environment (Fig. S17).
However, our DFT calculations provide a relative free energy of
-
-
the [Ga(L2)OH] species with respect to [Ga(L1)OH] of -11.3 kJ
-1
III
mol , reflecting again the ability of L2 to form more stable Ga
complexes.
Radiochemistry
The practical value of a given chelator for application in 68Ga-
PET radiopharmaceuticals is determined by two main aspects,
namely, its resistance against dissociation of the radiometal in
living organisms (sometimes referred to as “in vivo stability”)
Figure 6. 68Ga -incorporation as functions of concentration for L1 (a) and L2
(
3
III
b) for different pH values (reaction for 5 min at 25°C, mean values ± SD, n =
; incorporation was determined by radio-TLC). At 10 µM ligand concentration,
optimal labeling of both ligands is achieved at neutral to slightly basic pH (c).
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Chemistry - A European Journal
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differences are observed in a transchelation challenge against
aq. disodium EDTA. In order to enable observation over a larger
time period, these experiments were conducted with the long-
lived isotope 67Ga (t
.4, 37 °C). Fig. 8 shows that in comparison to the parent
= 3.3 d) under physiological conditions (pH
½
7
67
-
structure [ Ga][Ga(AAZTA)] , the annulated carbocycle causes
a strong destabilisation for [ Ga][Ga(L1)OH] but results in a
higher kinetic inertness of [ Ga][Ga(L2)OH].
67
6
7
Figure 7. 68Ga -incorporation vs concentration for L1 (left) and L2 (right) at
III
different temperatures (reaction for 5 min at pH 7.5, mean values ± SD, n = 3).
However, the cyclic polyaza-polycarboxylate chelators DOTA
and NOTA, which are most frequently used in 68Ga tracers, do
not allow for labelling at neutral pH, single-digit µM concentration,
and room temperature within a few minutes, while this was
found to be feasible for several acyclic structures at slightly
acidic
pH[38]
as
well
as
for
phosphinate-pendant
[
39,40]
Figure 8. Percentage of intact 67Ga -complexes in 50 mM aq. disodium EDTA
III
triazacyclononanes at pH 3.
Against this background, it is
solution as functions of time (37°C, pH 7.4, mean values ± SD, n = 3).
remarkable that the optimal pH for labelling of PIDAZTA isomers
L1/L2 lies between 7 and 8 while at pH 7.5, nearly quantitative
labelling (89.2% and 93.5% for L1 and L2, respectively) at room
temperature is achieved already at chelator concentrations
below 10 µM (Fig. 6).
Notably, elevated temperatures have an almost negligible
influence and do not substantially improve the labelling yield at
lower concentrations, particularly for L2 (Fig. 7). Such behaviour
might be considered counterintuitive because it is fundamentally
different from many established 68GaIII chelators, particularly
those based on polyazacycloalkanes, which exhibit the
corresponding pH-optimum at values around 3-4[39-41] and show
a strong influence of temperature on the required chelator
concentration (as a general rule, an approximately 30-times
In summary, the radiochemical investigations proved the
suitability of L2 for the elaboration of radiopharmaceuticals
based on gallium isotopes, combining highly efficient labelling
and strong resistance against demetallation at physiological
conditions.
Experimental
1. Materials
Ga(NO
6M HNO3 and evaporating of the excess acid. The solid
Ga(NO was dissolved in 0.1 HNO solution. The
concentration of the Ga(NO ) solution was determined by using
3 3 2 3
) was prepared by dissolving Ga O (99.9%, Fluka) in
lower concentration is required to achieve the same
radiolabelling yield at 95°C as compared to 25°C).[38] An
)
3
M
3
explanation might be that formation of L1/L2 complexes from
3
(OH)3n (the prevalent form of 68GaIII at neutral pH) and the
[42]
Ga
competing dehydration of this hydroxide, yielding non-reactive,
n
3 3
[
43]
2 2 2 2
standardised Na H EDTA in excess. The excess of Na H EDTA
[
44]
(“colloidal 68Ga”), are apparently
was measured with a standardized ZnCl2 solution and xylenol
orange as indicator. The concentration of CaCl (Sigma), MnCl
Sigma), ZnCl (Sigma), CuCl (Sigma) and LnCl solutions were
determined by complexometric titration with standardized
Na EDTA and xylenol orange (ZnCl and LnCl ), murexide
), Patton & Reeder (CaCl ) and eriochrome black T
) as indicator. The H+ concentration of the Ga(NO
insoluble [Ga(O)OH]
accelerated to a comparable extent upon heating (Scheme 3;
dk /dT ≈ dk /dT), essentially resulting in similar labelling yields
n
2
2
(
2
2
3
1
2
over a wide range of temperatures.
2
H
2
2
3
(
(
CuCl
MnCl
2
2
2
3
)
3
solution was determined by pH potentiometric titration in the
presence of excess Na
EDTA.[9] The concentration of
2
H
2
Scheme 3. Formation of 68Ga(L), 68Ga(OH)
and 68Ga(O)OH species.
3
PIDAZTA isomers (L1 and L2) and H AAZTA stock solutions
4
was determined by pH-potentiometric titrations in the presence
2
+
and absence of a 40-fold excess of Ca . The citrate solution
Apart from minor quantitative deviations, both isomers
nonetheless exhibit comparable overall labelling profiles.
However, in accordance with kinetic data (Table 2), fundamental
was prepared from H Citrate (Sigma) and its concentration was
determined by pH-potentiometry. The pH-potentiometric
titrations were made with standardized 0.2 M NaOH.
3
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Chemistry - A European Journal
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FULL PAPER
formed L1 and L2 were determined by pH-potentiometric
titration from acidic to basic pH range. The metal-to-ligand
concentration ratios were 1:1 (the concentrations of the ligands
were generally 0.002 M). The stability and protonation constants
2
. Single Crystal X-Ray Diffraction Analysis
After careful inspection of the crystallization batches at the
optical microscope, X-ray quality samples were selected,
resulting in colorless needle-like prisms with dimensions of
about 0.25 x 0.15 x 0.10 mm. The crystals of 3 and L1 were
III
of the “cold” Ga -L1/L2 complexes were calculated from the pH-
III
potentiometric titration of the Ga -L systems. The pH-
III
mounted on
a
Bruker AXS APEXII CCD area-detector
potentiometric titrations of Ga -L1 system were performed from
diffractometer, at room temperature, for the unit cell
determination and data collection. Graphite-monochromatized
MoKα (λ = 0.71073 Å) radiation was used with the generator
working at 50 kV and 30 mA. Orientation matrixes were initially
obtained from least-squares refinement on ca. 300 reflections
measured in three different ω regions, in the range 0° < θ < 23°;
cell parameters were optimized on the position, determined after
integration, of ca. 8000 reflections. The intensity data were
retrieved in the full sphere, within the θ limits reported in the cif
acidic to basic and from basic to acidic pH range by studying the
competition reaction between Ga and H+ for L1, and L1 and
III
-
III
III
-3
OH for Ga , respectively ([L]=[Ga ]=210 M). Because of the
high kinetic inertness, the stability and protonation constants of
Ga(L2) were calculated from the pH-potentiometric data
obtained from basic to acidic pH range, whereas the protonation
constants of Ga(L2) were also evaluated from the pH-
potentiometric titration achieved from acidic to basic pH range
III
-3
-
([L]=[Ga ]=210 M). The protonation constants of [Cu(L1)] and
-
files, from 1080 frames collected with
a
sample−detector
distance fixed at 5.0 cm (50 s frame ; ω scan method, Δω =
.5°). An empirical absorption correction was applied
SADABS).[45] Crystal structure was solved by direct methods
[Cu(L2)] were determined by pH-potentiometric titrations of CuL
−
1
complex in the pH range of 1.7–11.7 ([CuL]=210-3 M). For pH
measurements and pH-potentiometric titrations, a Metrohm 785
DMP Titrino titration workstation and a Metrohm-6.0233.100
combined electrode were used. The pH potentiometric titrations
were performed at constant ionic strength (0.15 M NaCl) in 6 mL
0
(
using SHELXT2017 and refined with SHELXL-2017/1[46,47] within
the Wingx suite of programs.[48] Hydrogen atoms were riding on
their carbon atoms, for 3 and, for L1, were freely refined on their
positions, derived from the difference Fourier map. Anisotropic
temperature factors were assigned to all non-hydrogen atoms.
Crystal data collection and refinement parameters are listed in
the supporting information and in the cif files. A view of the
molecules with the full numbering scheme is given in Fig. S1
and S3. Selected distances of bond lengths (Å) and angles (°)
are given in Table S1 and S2, while atomic coordinates and
displacement parameters are listed in the corresponding cif file.
CCDC numbers 1872753 and 1872767 contain the full
supplementary crystallographic data for this work. The latter can
be obtained free of charge from the Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
samples at 25 C. The solutions were stirred, and N
2
was
bubbled through them. The titrations were made with 300 s
waiting time in the pH range of 1.7-11.7. KH-phthalate
(pH=4.005) and borax (pH=9.177) buffers were used to calibrate
+
the pH meter. For the calculation of [H ] from the measured pH
values, the method proposed by Irving et al. was used.[49]
A
0.01M HCl solution was titrated with the standardised NaOH
solution in the presence of 0.15 M NaCl ionic strength. The
differences between the measured (pHread) and calculated pH (-
log[H+]) values were used to obtain the equilibrium H+
concentration from the pH values, measured in the titration
experiments. The ionic product of water (pK
w
) at 25 C in 0.15 M
-
NaCI was found to be 13.83. The stability constant of [Cu(L1)]
-1
-
+
Crystal data for 3: C17
P2 /c (No. 14), a = 7.8915(4), b = 23.0741(13) and c = 9.3186(5)
Å, β = 93.0160(10) °; V = 1694.47(16) Å3 ; Z = 4; Mo-K
λ =
) = 0.079
H
25
N
3
O
2
, fw = 303.4 gmol , monoclinic
and [Cu(L2)] was determined by spectrophotometry in the [H ]
2
+
-3
1
range of 0.01-1.0 M ([L]=[Cu ]=110 M). Seven samples were
+
+
α
prepared and the H concentration ([H ]=0.010, 0.025, 0.050,
0.10, 0.35, 0.60 and 1.0 M) in the samples was adjusted with the
addition of calculated amounts of 2.0 M HCl. The samples were
kept at 25 C for 7 days in order to attain the equilibrium (the
time needed to reach the equilibrium was determined by
spectrophotometry). The absorbance values of the samples
were measured at 11 wavelengths (575, 595, 615, 635, 655, 675,
695, 715, 735, 755 and 775 nm). The ionic strength of samples
-
3
0
.71073 Å; T (K) 293(2); ρcalc = 1.189 g cm , μ(Mo-K
α
-1
mm ; θ range 1.765-26.439 °; data (unique), 3473 (2965);
restraints, 0; parameters, 202; Goodness-of-Fit on F , 1.035; R
2
1
and wR
2 1 2
(I>2σ(I)), 0.0485 and 0.1203; R and wR (all data),
–
3
0.0555 and 0.1259; Largest Diff. Peak and Hole (e Å ), 0.246
and -0.186.
27 3 6
Crystal data for L1 (4R*,10aS*)-PDAZTA: C16H N O , fw =
-
1
+
3
7
1
=
57.4 gmol , monoclinic P2
1
/c (No. 14), a = 9.8286(5), b =
with [H ]=0.32, 0.60 and 1.0 M was not constant (the ionic
+
.9931(4) and c = 21.3905(10) Å, β = 93.1966(8) °; V =
677.84(27) Å ; Z = 4; Mo-K
1.415 g cm , μ(Mo-K
strength of samples with [H ]=0.010, 0.025, 0.050, 0.10 M was
3
+
+
α
λ = 0.71073 Å; T (K) 293(2); ρcalc
[H ]+[Na ]=0.15 M). For the equilibrium calculations, the molar
-3
-1
II
α
) = 0.108 mm ; θ range 1.907-26.525 °;
absorptivities of the Cu , CuL, CuHL and CuH
2
L species were
II
-
-
data (unique), 3473 (2999); restraints, 0; parameters, 335;
used. The molar absorptivities of Cu , [Cu(L1)] and [Cu(L2)]
2
Goodness-of-Fit on F , 1.038; R
1
and wR
2
(I>2σ(I)), 0.0373 and
complexes were determined by recording the Vis spectra
(=400-800 nm) of 1.010 , 2.010 , 3.010 and 4.010-4
M
-
4
-4
-4
0
.0957; R
1
and wR
2
(all data), 0.0434 and 0998; Largest Diff.
–
3
Peak and Hole (e Å ), 0.249 and -0.174.
solutions in the pH range 1.7-7.0 (0.15 M NaCl, 25 C). The pH
was adjusted by stepwise addition of concentrated NaOH or HCl.
The spectrophotometric measurements were made with a Cary
1E spectrophotometer at 25 C, using 1.0 cm cells. The
3. Equilibrium measurements
The protonation constants of L1 and L2, and the stability and
II
II
II
III
protonation constants of Ca -, Mn -, Zn - and Ln -complexes
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Chemistry - A European Journal
10.1002/chem.201901512
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protonation and stability constants were calculated with the
PSEQUAD program.[50]
and 20 times higher, to ensure pseudo-first-order conditions. In
order to prevent the hydrolysis of GaIII and CuII ions, the
transmetallation reactions were studied in the presence of citrate
4
. NMR experiments
1
H- and 71Ga-NMR measurement were performed with a Bruker
DRX 400 (9.4 T) instrument equipped with a Bruker VT-1000
thermocontroller and a BB inverse z gradient probe (5 mm). The
formation and protonation/deprotonation processes of the
t
excess ([Cit] =2.0 mM). The exchange rates were studied in the
pH range about 6.0-9.0. For keeping the pH values constant,
MES (pH range 6.0-7.0), HEPES (pH range 7.0-8.5) and
piperazine (pH range 8.5-9.0) buffers (0.01 M) were used. The
temperature was maintained at 25 C and the ionic strength of
the solutions was kept constant (0.15 M NaCl). The pseudo-first-
[
Ga(L1)] and [Ga(L2)] were followed from basic to acidic pH
range at 298 K in 0.15 M NaCl. For these experiments, 9.3 mM
and 9.5 mM solutions of the [Ga(L1)] and [Ga(L2)] complexes in
d
order rate constants (k ) were calculated from the slope of the
H
2
O were prepared, respectively (A capillary with D
2
O was used
absorbance vs. time curves (Abs/t) with Eq. 10 in Supporting
Information. For the calculations, the molar absorptivities of
[Cu(L1)] , [Cu(L2)]- and Cu(Cit)H-1 were used, which were
for lock). The pH was adjusted with the addition of concentrated
solutions of NaOH or HCl. Because of the metal exchange
between the [Ga(L1)OH] or [Ga(L2)OH] and [Ga(OH)
ligand exchange between the complexes and the free ligand
were in the “slow exchange regime” on the actual NMR
-
-
-
-4
4
] and the
determined at 300 nm by recording the spectra of 1.010 ,
-
-4
-4
-4
2.010 , 3.010 and 4.010 M solutions in the pH range 5-10
(0.15M NaCl, 25C). The calculations were performed with the
use of the computer program Micromath Scientist, version 2.0
(Salt Lake City, UT, USA).
timescales, the calculation of the logGa(L)OH value of
Ga(L1)OH]- and [Ga(L2)OH] was performed by using the
-
[
intensities of the 71Ga-NMR signal of [Ga(OH)
] complex and
-
6. Trans-chelation reactions with human serum transferrin
4
the 1H-NMR signal of the –CH
[
[
protons in [Ga(L1)OH] and
-
The ligand exchange reaction between [Ga(L1)OH] ,
-
3
Ga(L2)OH] . The molar intensity values of 71Ga-NMR signal of
Ga(OH) ] complex and the H-NMR signal of the –CH protons
4
3
-
[Ga(L2)OH] and human serum transferrin (Sigma, partially Fe -
saturated) have been studied by spectrophotometry, following
the formation of Ga(sTf) complex at 246 nm and pH=7.4 with the
use of 1.0 cm cells and Cary 1E spectrophotometer. The
concentration of the human serum transferrin solution was
-
III
-
1
-
-
in [Ga(L1)OH] and [Ga(L2)OH] complexes were determined by
recording the H-NMR spectra of 0.01, 0.015, 0.02 and 0.025 M
solutions of [Ga(L1)OH] and [Ga(L2)OH] complexes at pH=6.0
and Ga-NMR spectra of 0.01, 0.015, 0.02 and 0.025 M
1
-
-
7
1
determined from the absorbance at 280 nm using the molar
-
-1 -1 [51]
solutions of [Ga(OH)
4
] at pH=12.5 (0.15 M NaCl, 25 C). The
absorptivity

280=91200 cm M .
In order to ensure the
formation of [Ga(L1)] and [Ga(L2)] complexes were also studied
pseudo-first-order condition, the rate of the ligand exchange
1
71
+
III
by H- and Ga-NMR spectroscopy in the [H ] range 0.01-1.00
reactions were studied in the presence of high excess of Ga -
+
-
-
M. In these experiments, 6+6 samples were prepared ([H ]=0.01, complexes ([Ga(L1)OH] =[Ga(L2)OH] =0.1 and 0.2 mM, [sTf]=10
0
.03, 0.13, 0.25, 0.50 and 1.0 M) for [Ga(L1)] and [Ga(L2)]
M)). The temperature was maintained at 25C, the ionic
strength and the hydrogen-carbonate concentration of the
samples were kept constant; 0.15 M for NaCl and 0.025 M for
complexes in H O (a capillary with D O was used for lock). The
concentrations of [Ga(L1)] and [Ga(L2)] complexes were 9.3
mM and 9.5 mM. The [H ] was adjusted with the addition of
calculated amounts of 2.0 M HCl. The samples were kept at 25
2
2
+
NaHCO
7. Computational details
Geometry optimizations
[Ga(L1)OH] and [Ga(L2)(OH)] systems were performed using
DFT calculations at the TPSSh/TZVP
3
, respectively.
C for 7 days in order to attain the equilibrium. The ionic strength
of
-
the
[Ga(L1)],
[Ga(L2)],
+
-
of samples with [H ]=0.25, 0.50 and 1.0 M was not constant (the
ionic strength of samples with [H ]=0.01, 0.03 and 0.13 M was
+
[52,53]
level with the
+
+
[54]
[
H ]+[Na ]=0.15 M). Because of the metal exchange between
Gaussian 09 package (Revision D.01). Solvent effects (water)
were included by using the polarizable continuum model (PCM),
in which the solute cavity is built as an envelope of spheres
centered on atoms or atomic groups with appropriate radii.
Specifically, we used the integral equation formalism variant of
the polarizable continuum model (IEFPCM) as implemented in
Gaussian 09.[55] No symmetry constraints were imposed during
the optimizations. The stationary points found on the potential
energy surfaces as a result of geometry optimizations were
confirmed to correspond to energy minima rather than saddle
points using frequency calculations. Wave function analysis was
carried out by computing the electron density () at the bond
the [Ga(L1)] and [Ga(L2)] and the free GaIII ion was in the “slow
exchange regime” on the actual NMR timescale, the calculation
of the logKGaL value of [Ga(L1)] and [Ga(L2)] was also
performed with the use of the 71Ga-NMR intensities of Ga ion
by taking into account the protonation constants of the [Ga(L1)]
and [Ga(L2)] complexes. The molar intensity value of 71Ga-NMR
signal of GaIII ion was determined by recording the 71Ga-NMR
III
III
spectra of 0.01, 0.015, 0.02 and 0.025 M solutions of Ga in the
presence of 1.0 M HNO
3
. Calculation of the logKGaL and
+
logGa(L)OH values was performed by using the integral-[H ] data
pairs with the PSEQUAD program.[50]
[
56]
5
. Trans-metallation reactions
The rates of the exchange reactions taking place between
Ga(L1)], [Ga(L2)] and CuII in the presence of citrate were
studied by spectrophotometry, following the formation of the
critical points (BCP) with the computer program Multiwnf 3.2.
3
+
Geometry optimizations of the systems [Ga(H
2
O)
6
] ·12H
2
O,
[
[Ga(OH)
4
]-·8H
2
O, [Ga(NOTA)] and [Ga(DATA )] and subsequent
m
frequency calculations were performed using the same
methodology.
The calculations of the 71Ga NMR shielding tensors were
carried out using the ORCA program package (Version 3.0.1)[57]
-
-
[
Cu(L1)] and [Cu(L2)] complexes at 300 nm, with the use of 1.0
cm cells and a Cary 1E spectrophotometer. The concentration of
Cu was 0.1 and 0.2 mM, while that of Ga -complexes were 10
II
III
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Chemistry - A European Journal
10.1002/chem.201901512
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using the TPSSh functional and the own nucleus as the gauche
origin. Relativistic effects were included with the second order
Douglas-Kroll-Hess (DKH2) method[58,59] in combination with the
all-electron scalar relativistic TZVPPP-DKH basis set.[60] The
RIJCOSX approximation[61-64] was used to accelerate the
which particularly rapidly decomposes in a transchelation
challenge vs EDTA, the (4R*,10aS*)-isomer does not show any
substantial release of the radiometal under these conditions.
However, both isomers quantitatively incorporate 68GaIII within
minutes at room temperature and chelator concentrations below
10 M. This occurs most effectively at pH values between 7 and
8. PIDAZTAs are therefore the first chelators reaching their
optimal labeling performance at truly physiological conditions.
The demetallation-resistant isomer, (4R*,10aS*)-PIDAZTA, is
therefore perfectly suited for use in combination with thermo- or
acid-sensitive biomolecules. However, more importantly,
PIDAZTA-based compounds appear ideal for application in
68Ga-labelling kits. This is because the labeling reaction can be
carried out at physiological pH (7.4) which is demanded for
parenteralia in most pharmacopoeia documents, thus obviating
addition of buffers after labelling or making compromises in
terms of product specification. It is anticipated that clinical
preparation of 68Ga radiopharmaceuticals will be dominated by
[
65]
calculations with the aid of the Def2-TZVPP/JK auxiliary basis
set. The SCF convergence tolerances and integration
accuracies of the calculations were increased from the defaults
using the available TightSCF and Grid5 options (Grid7 for Ga).
Solvent effects (water) were considered by using the COSMO
solvation model as implemented in ORCA.[66] Chemical shifts
were calculated as  = (isoref-iso) using [Ga(H
6 2
O) O as
]3+·12H
2
a reference. The iso values can be broken down into the
d
p
diamagnetic ( ) and paramagnetic ( ) contributions, which
provide shielding and deshielding contributions, respectively
(
Table S7).[67,68]
8
A
. Radiochemistry
6
8
68
Ge/ Ga-generator with SnO
2
matrix (obtained from
©
ITHEMBA LABS, South Africa) was eluted with 1.0 M aq. HCl. A
fraction of 1.25 mL containing the highest activity (ca. 400 MBq)
was mixed with a solution of HEPES (2 M; 0,800 µL) or sodium-
acetate (4 M; 0.8 µL). From this solution aliquots of 90 µL
labeling kits in the near future. One such kit, Somatokit-TOC ,
has received a marketing authorisation recently, and many more
are currently in the approval pipeline of several companies.
Hence, we conclude that PIDAZTA represents a valuable
building block for future development of advanced 68Ga radio-
pharmaceuticals.
(
containing about 15 MBq or 0.15 pmol each) were transferred
into eppendorf cups. 10 µL of stock solutions of the ligand
(
(
ranging 0.3 µM – 100 µM) were added and mixed well. Heating
if applied) was performed by placing the closed eppendorf vials
into a thermostated water bath. After heating, labeling reactions
were interrupted by placing the cups into a cold-water bath.
Acknowledgements
Samples were analyzed by TLC (1.0 M NH
mobile phase, where insoluble colloidal 68GaIII stays at the origin
= 0) and the radiolabeled product is eluted with the solvent
front (R = 0.5-0.6). The adjustment of different pH values for
4
OAc/MeOH (1:1) as
Authors C. P.-I and D. E.-G. thank Centro de Supercomputación
de Galicia (CESGA) for providing the computer facilities. VC
thanks Prof. A. Sironi for fruitful discussions and University of
Milan for partial funding (PSR2018). Authors are grateful for the
support granted by the Hungarian National Research,
Development and Innovation Office (NKFIH K-128201 project).
The research was also supported in a part by the EU and co-
financed by the European Regional Development Fund under
the projects GINOP-2.3.2-15-2016-00008 and GINOP-2.3.3-15-
(R
F
F
assessment of pH-dependence was completed with further
addition of 1 M aq. NaOH or 1 M aq. HCl to the labelling solution
6
7
prior to addition of the ligands. Synthesis of Ga complexes was
performed likewise, using 67Ga in 0.05 M HCl (obtained from
Mallinckrodt, Petten, The Netherlands) instead of 68Ga.
67
Stabilities of the [ Ga][GaL] complexes were tested by adding
00 µL (1 mM) of the product to 900 µL of 100 mM EDTA
2016-00004.
1
solution, followed by incubation at 25 °C for 5 d. Percentages of
intact complexes were quantified using radio-TLC, by
withdrawing samples (2.5 µL) during the reaction at 30, 60 and
Conflicts of interest
9
0 min and spotting onto the TLC strip.
There are no conflicts of interest to declare.
Conclusions
A conformational locking of mesocyclic diazepine-type chelators
was achieved by the annulation of an additional six-membered
saturated carbocycle to the 6-amino-1,4-diazepane moiety.
Since the fused piperidine cycle encompasses one of the ring
nitrogens, the resulting piperidino[1,2-a]diazepine-triacetic acid
Keywords: gallium • chelating agent • stability •
thermodynamics • kinetics
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Chemistry - A European Journal
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10.1002/chem.201901512
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E. Farkas, A. Vágner, R. Negri, L.
Lattuada, I. Tóth, V. Colombo, D.
Esteban-Gómez, C. Platas-Iglesias, J.
Notni,* Zs. Baranyai,* G.B. Giovenzana*
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PIDAZTA: Structurally Constrained
Chelators for Efficient Formation of
Stable Gallium-68 Complexes at
Physiological pH
PIDAZTAs: bicyclic ligands for the development of 68Ga-based
radiopharmaceuticals. Quantitative radiolabelling in mild conditions in minutes and
stereochemistry-driven thermodynamic and kinetic stability.
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