Investigation of the complexation ofnatZr(iv) and89Zr(iv) by hydroxypyridinones for the development of chelators for PET imaging applications

Article abstract of DOI:10.1039/c6dt04625h

Three hydroxypyridinone (HOPO) positional isomers-1,2-HOPO (L¹H) and its water soluble analogue (L^1′H), 3,2-HOPO (L²H) and 3,4-HOPO (L³H) have been investigated for the complexation of Zr(iv). Potentiometric and

Full text of DOI:10.1039/c6dt04625h

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ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
rsc.li/dalton

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Investigation of the complexation of natZr(IV) and 89Zr(IV) by
hydroxypyridinones for the development of chelators for PET
imaging applications
Received 00th January 20xx,
Accepted 00th January 20xx
F. Guérarda,b*, M. Beylerc, Y.-S. Leed, R. Tripierc, J.-F. Gestina and M. W. Brechbielb
DOI: 10.1039/x0xx00000x
Three hydroxypyridinones (HOPOs) positional isomers - 1,2-HOPO (L1H) and its water soluble analogue (L1’H), 3,2-HOPO
(L2H) and 3,4-HOPO (L3H)- have been investigated for the complexation of Zr(IV). Potentiometric and UV-Vis spectrometric
studies show a higher thermodynamic stability for the formation of Zr(L1')4 in comparison with Zr(L2)4 and Zr(L3)4 as well as
a higher kinetic inertness in competition studies with EDTA or Fe3+ at radiotracer concentration with 89Zr. Besides the low
pKa of L1H or L1’H (pKa = 5.01) in comparison with L2H and L3H (pKa = 8.83 and 9.55, respectively), the higher stability of
Zr(L1')4 can be attributed in part to the presence of the amide group next to the chelating oxygens that induces
intramolecular H-bond and amide/π interactions that were observed by X-ray crystallography and confirmed by quantum
chemical calculations. The data presented here indicates that the 1,2-HOPO L1' exhibits the best characteristics for Zr(IV)
complexation. However, 2,3-HOPO and 3,4-HOPO patterns, if appropriately tuned, for instance with the addition of an
amide group as in the 1,2-HOPO ligand, may also become interesting alternatives for the design of Zr(IV) chelators with
improved characteristics for application in nuclear imaging with 89Zr.
www.rsc.org/
imaging.6–8
The Zr(IV) cation, with its relatively small ionic radius (0.84 Å)
and high positive charge number, exhibits the characteristics
Introduction
Hydroxypyridinones (HOPOs) are compounds that have long
been studied for their ability to complex hard metallic cations
via their oxygen atoms (Scheme 1).1 They have particularly
been considered as medical tools for complexation of Fe(III)
for the treatment of iron overload.2,3 Due to the electronic
properties of actinides (IV) elements being similar to Fe(III),
HOPO-based chelators have also been developed as
sequestering agents of actinides for decontamination or
decorporation applications.4 HOPOs have also recently proven
useful for the stable complexation of Gd(III) associated with
improved relaxivity and sensitivity of Gd-based contrast agents
for magnetic resonance imaging (MRI).5 In the field of nuclear
medicine, HOPOs have also been studied as ligands of Ga(III),
whose radioisotopes 67Ga and 68Ga can be used for nuclear
of a hard Lewis acid and is ideal for strong complexation by
hard bases such as HOPOs. In recent years, this metal has
received a growing interest in nuclear imaging of cancers with
the use of the β+-emitter 89Zr. With its relatively long half-life
(t1/2 = 78.4 h) and favorable positron energy (βavg = 395.5 keV),
high tumor/background signal ratio can be acquired up to
several days after administration to the patient when 89Zr is
associated with a long blood circulating vector such as an
antibody. This radionuclide broadens the panel of β+-emitters
available (68Ga, t1/2 = 1.1 h; 18F, t1/2 = 1.8 h; 44Sc, t1/2 = 3.97 h;
64Cu, t1/2 = 12.7 h; 86Y, t1/2 = 14.7 h or 124I, t1/2 = 100.2 h as
examples of the most studied to date),9–11 and expands the
range of diseases that can be imaged by positron emission
tomography (PET).
To date, the only chelating agent that has proven successful
for labeling biomolecules for clinical studies with 89Zr is
desferrioxamine B (DFB, see Scheme 1), a natural siderophore
comprised of three hydroxamic acid subunits involved in the
complexation of Zr(IV).12 A large number of antibodies has
been labeled with 89Zr-DFB and used in pre-clinical and clinical
studies in recent years.13 However, in a number of cases, the in
vivo stability of the Zr-DFB complex has proven insufficient as
seen by the accumulation of free, osteophilic 89Zr in bones 2 to
4 days after injection of the labeled antibody.14–17
a.CRCNA, INSERM, CNRS, Université d'Angers Université de Nantes, Nantes, France.
E-mail: francois.guerard@univ-nantes.fr
b.Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch,
National Cancer Institute, National Institutes of Health, Bethesda, Maryland,USA
c. Université de Bretagne Occidentale, UMR-CNRS 6521 CEMCA, UFR des Sciences et
Techniques, Brest, France
d.Center for Molecular Modeling, Division of Computational Bioscience, Center for
Information Technology, National Institutes of Health, Bethesda, Maryland, USA
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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the tumor which indicates that further improvements are still
necessary.34
Whereas the development of HOPO-based chelators appears
to be a viable alternative to hydroxamates, surprisingly little is
known about the aqueous structure and thermodynamic
stability of such Zr-HOPO complexes. This partial vision of the
aqueous chemistry of Zr(IV) is a limitation for the rational
design of optimally pre-organized chelating agents based on
this class of ligands. Accordingly, we have initiated an in-depth
investigation on the complexation chemistry of Zr(IV) towards
the 1-hydroxy-2(1H)-pyridinone (1,2-HOPO) unit as well as its
structural isomers 3-hydroxy-2(1H)-pyridinone (3,2-HOPO) and
3-hydroxy-4(1H)-pyridinone (3,4-HOPO) (Scheme 1), differing
in their ability to sequester Zr(IV).
Results and discussion
Scheme 1. Structure of ligands discussed in this article.
Synthesis of ligands and Zr(IV) complexes.
As models for the study, we chose to synthesize the most simple
and unconstrained HOPOs while introducing a short chain that
would mimic the electronic density on the ring when attached to
the backbone of a chelator. A rich synthetic chemistry of HOPOs is
available from Raymond or Hider's groups which have developed an
array of HOPO-based chelators. In most cases, convenient methods
were to introduce a carboxylic acid on the ligand structure for
conjugation to polyamines with formation of amide bonds.2,35–38
Accordingly, the O-benzyl protected HOPOs were synthesized
following previously reported procedures.35,39,40 After conversion
into their N-hydroxysuccinimidyl esters, they were conjugated to
methylamine and deprotected to form 1,2-HOPO-NHMe (L1H), 3,2-
HOPO-NHMe (L2H) and 3,4-HOPO-NHMe (L3H). Both L2H and L3H
were soluble in water whereas L1H exhibited poor solubility in this
solvent. This lower solubility may be attributed to intramolecular
hydrogen bonding of the amide hydrogen with the 1-hydroxy group
in L1H that lowers the ability of the molecule to be solvated by
water via hydrogen bonding, as reported with similar compounds.37
Consequently, a variation of L1H was also synthesized (L1'H), bearing
a n-propyl instead of the methyl and reported to be water soluble.41
The improved water solubility made then possible further aqueous
stability studies (Scheme 2). It is worth noting that besides the
difference in the hydroxyl group position, a striking feature of (L1H)
or (L1'H) that (L2H) and (L3H) do not exhibit is the presence of an
amide group directly connected to the ring on the carbon next to
the nitrogen. This amide group is expected to greatly influence
metal complexation by modification of the electron density on the
hydroxyl by inductive effect, and the pKa by stabilization of the
deprotonated form due to hydrogen bonding as previously
observed in other HOPOs studies.37,42 Zr(HOPO)4 complexes were
formed by transchelation of zirconium(IV) acetylacetonate in the
presence of a slight excess of ligand (4.5 equiv.) in refluxing
methanol. All ligands formed the expected ZrL4 complexes as
confirmed by NMR, mass spectrometry and elemental analyses.
After more than 15 years of imaging studies with the sub-
optimal 89Zr-DFB complex, only very recently have chemistry
groups initiated research programs to provide new chelating
agents with improved characteristics for stable complexation
of 89Zr(IV) and safer in vivo use and improved sensitivity.
Recent studies have provided a better insight of the aqueous
behavior of Zr(IV) in the presence of hydroxamate ligands by
crystallographic, potentiometric and quantum chemical
studies.18,19 Results indicate the tendency of Zr(IV) to form a
more stable complex in the presence of an octadentate
chelator as compared to the hexadentate DFB. Accordingly,
several octadentate ligands have been proposed to fit the
Zr(IV) requirements. Most of the investigations have focused
on the development of chelators bearing hydroxamate groups
with either the acyclic,20–22 the cyclic23 or the aromatic
hydroxamate (1,2-HOPO) pattern.24 These recent advances
have led to the first hydroxamate based bifunctional chelators
that provide 89Zr complexes with improved in vivo stability
compared to DFB25,26
.
Alternatively, chelators bearing
catechols,27,28 phosphonates29 or 3,2-HOPOs30 have also been
proposed. The development of HOPO based chelators for
nuclear imaging application is not new and several examples of
chelators for 67Ga or 68Ga complexation have been reported
7,8,31
over the recent years.
In line with these reported
applications, bifunctional HOPO based chelators for 89Zr
complexation have recently been developed and studied in
vivo with varying degrees of success.30,32,33 The most stable to
date, p-SCN-Bn-HOPO which is based on the 1,2-HOPO
subunit, released a significantly lower fraction of the 89Zr
activity to the bone in comparison with the same antibody
radiolabeled using DFB-SCN. This new compound is however
accompanied by a significant decrease of the 89Zr activity in
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Scheme 2. Synthesis of HOPOs L L L
1H, 2H and 3H. i) EDCI, NHS, DMF, ii) R-NH2, THF, iii) AcOH/HClconc (1:1), iv) H2 (30 psi),
Pd/C (10%), MeOH, v) Zr(acac)4, MeOH. L1'H was prepared in accordance with the reported procedure.41
Additionally, Zr(L1)4 and Zr(L2)4 were isolated as crystals suitable for the ligands in Zr(L1)4 are oriented as two pairs of stacked rings
X-ray diffraction studies.
resulting in the HOPO rings being significantly pushed out of the
planes formed by Zr and its chelating oxygen atoms (Figure 1c). For
instance, plane (a) formed with Zr and its pair of chelating oxygen
atoms (O4 and O5) intersects the ring plane (b) with an angle of 25°
whereas it is only 10° in Zr(L2)4 (Figure 1c). Such a geometry can be
Structure elucidation by crystallography and quantum chemical
calculations.
To optimally design pre-organized chelators with HOPOs, it is
essential to know how the free ligands organize themselves without
external constraint when forming a complex with the Zr4+ cation.
Single crystal crystallography (see experimental details in ESI) shows
that both L1 and L2 form a complex in which the central Zr atom is
octacoordinated by four hydroxypyridinonates via their oxygen
atoms (Figure 1a & 2). Zr(L2)4 differs by a lower degree of symmetry
(triclinic crystal system) than Zr(L1)4 (monoclinic). Whereas each
pair of oxygen atom exhibits a high symmetry in Zr(L1)4 (i.e Zr-O1 =
2.209 Å and Zr-O2 = 2.170, all Zr-O comprised between 2.165 and
2.215 Å), it is significantly lower in the case of Zr(L2)4 (Zr-O distances
between 2.127 and 2.305 Å). Crystals of Zr(L3)4 could not be
obtained and thus we resorted to quantum chemical calculation to
obtain structural information of Zr(L3)4. This structure was built by
modifying the crystal structure of Zr(L1)4 and then geometry
optimized in the reaction field of water. The calculated structure is
analogous to Zr(L2)4 with Zr-O distances ranging from 2.187 to 2.335
Å (Figure S4). A distinctive feature of the Zr(L1)4 complex is the H-
bonds formed between the amide hydrogen atoms and the oxygen
attributed to amide/heteroarene
π stacking, with the amide
nitrogen to HOPO ring center distance of 3.30 Å (Figure 1d) that
matches the calculated distance for similar interaction reported
between N-methylacetamide and pyridinone, providing additional
stabilization of the complex.43
atoms coordinating Zr with i.e (N2)H-O1
= 1.978 Å and a
donor/acceptor distance N2-O1 of 2.661 Å (Figure 1b). Such
intramolecular H-bonds are known to provide pre-organization and
subsequent extra stabilization to the complex as reported with
other metals such as Gd3+ 42
Additionally, whereas the four HOPO
.
rings are equally spaced around the Zr atom in Zr(L2)4 and Zr(L3)4,
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Figure 1. a) Crystal structure of Zr(L
1)4. Selected bond lengths (Å) and angles (°): Zr1-O1, 2.209; Zr1-O2, 2.170; O1-Zr1-O1,
69.73; N1-O1-Zr1, 113.49; C1-O2-Zr1, 117.68. b) View of H-bond between amide proton and adjacent oxygen donor. c)
Representation of the angle formed between the plane of Zr1-O4-O5 and the HOPO ring. d) View of Amide/HOPO ring π
stacking. Water molecules and hydrogen atoms omitted for clarity. Crystallographic summary available in ESI, Table S1.44
Thermodynamic stability studies.
To further study the properties of the HOPO moieties and their
affinity for Zr(IV), their acid-base properties and the complexation
with Zr(IV) were studied by potentiometric or UV-vis spectroscopic
titrations in aqueous solution. The measured constants were
compared with N-methyl-acetohydroxamic acid (Me-AHA, Scheme
1) which is the bidendate unit of DFB that we studied previously
and which is known to form very stable zirconium (IV) complexes.18
Ligand protonation constants were determined by potentiometric
titration carried out from low to high pH (results in Table 2). L1’ and
L2 protonation constants were determined to be log Ka = 5.01 and
log Ka = 8.83, respectively. L3 exhibits two successive protonation
steps with a first log Ka value of 9.55 and a second one of 2.99 that
were attributed to the 4-oxo group and the 3-hydroxy group,
respectively, as reported in similar compounds.37 These data show
that L1’ is significantly more acidic, which can be explained by the
position of the hydroxyl on the pyridinonate nitrogen and by the
Figure 2. Crystal structure of Zr(L
2)4. Selected bond lengths
adjacent amide group that acts on the acidity by two effects: i) the
electron withdrawing effect of the amide group and ii) the
intramolecular H-bond formed by the amide hydrogen that
stabilizes the deprotonated form of the ligand, two features that L2
and L3 do not exhibit. All protonation constants are in good
agreement with those previously reported by Raymond et al.41
(Å) and angles (°): Zr1-O1, 2.272; Zr1-O2, 2.127; O1-Zr1-
O1, 71.75; C1-O1-Zr1, 114.37; C2-O2-Zr1, 120.87. Water
molecules and hydrogen atoms omitted for clarity.
Crystallographic summary available in ESI, Table S1.44
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Table 1. Overall (log
with Zr4+ in aqueous solution at 25.0 °C. Literature data for Me-AHA are provided for comparison.15
β
) and stepwise (log K) thermodynamic protonation constants of L1’, L2 and L3 and their stability constants
L1’ a
L2 b
L3 b
Me-AHA c
log K
log β
Species
LHd
LH2
log K
5.01
-
log K
8.83
-
log K
9.55
2.99
log
β
log
β
log
β
5.01 (7)e
-
8.83 (4)
9.55 (3)
12.54 (4)
8.75
8.75
-
-
-
ZrL3
ZrL3H
ZrL4
31.3 (1)f
40.2 (1) f
49.40 (1) f
41.97 (1) f
31.3
8.9
18.1
-7.43
-
-
36.0 (6)
-
41.1 (3)
43.0 (4)
49.27 (5)
-
41.3
2.4
8.7
-
28.66 (5)
3.44
36.0 (6)
45.5 (1)
35.9 (1)
-
-
17.32
-
45.98 (1)
-
ZrL4(OH)
-9.6
c
18
a 25.0 °C, I = 0.1 M in KCl; b 25.0 °C, I = 0.2 M in KNO3; I = 0.1 M KNO3, previously reported data, see Guérard et al.
;
d Charges
are omitted for clarity. e Values in parentheses are standard deviations in the last significant digit; f Constants determined by UV
spectrophotometric titration.
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
ZrL3
c)
ZrL4
ZrL4
b)
a)
ZrL
ZrL44
ZrL H
ꢀ
4
ZrL4(OH)
1
ZrL3(OH)
Zr(OH)
ZrH
ꢀ
4
4
ZrL4(OH)
Zr(OH)3
Zr(OH)4
2
4
6
8
10
2
4
6
8
10
2
4
6
8
10
pH
pH
pH
Figure 3. Speciation diagrams of Zr species with: (a)
L
1’, (b) 2 and (c) L3
L .
Based on the X-ray structures of the complexes indicating the
favored formation of 1:4 (M:L) adducts, 0.25 equivalents of Zr(IV)
relative to the ligand was used during complexation titrations.
Measurements of the stability constants for L2 and L3 were
performed by potentiometry. For L1' whose Zr(IV) complexes
Table 2. Calculated p(Zr4+)a values for the complexes of L1’,
L2, L3 and Me-AHA.
L1'
33.48
L2
28.54
L3
28.64
Me-AHA
28.52
pZr4+
a Values calculated at pH = 7.4 with [Zr4+]tot = 1 × 10−5
M
exhibited
poor
aqueous
solubility,
we
resorted
to
and [L] = 5 x 10-5 M, based on the constants given in
Table 2.
spectrophotometric titration that allows working at lower
concentration than potentiometric methods and which prevented
precipitation issues. L1' complexes were monitored by a shift of the
absorbance from 315 nm to 345 nm. All results are reported in
Table 1.
All three HOPO ligands exhibit high stability of Zr(IV) complexes
with log KZrL4 > 45, but L1' and L3 (log KZrL4 = 49.4 and 49.27,
respectively) are superior to L2 and Me-AHA by four orders of
magnitude (log KZrL4 = 45.5 and 45.98, respectively). Interestingly,
despites its lowest log Ka, L1' exhibits the highest log KZrL4. This
observation deviates from the trends observed within other
reported HOPO series in which the cumulative stability constants
for metal complexation increase as proton affinity increases;
although some exceptions are observed (but not as marked as in
this study).37,38 This deviation from the trend with L1' may be
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explained in part by the extra-stabilization by H-bond and amide/π
stacking observed in the crystal structure (see previous section).
For a better comparison of Zr(IV) affinity independent of log Ka
values, pZr4+ values were calculated for each ligand at physiological
pH (Table 2). Results were nearly identical for L2, L3 and Me-AHA
(pZr4+ ≈ 28.5) whereas L1' exhibited a significantly higher pZr4+ value
of 33.48, confirming the better ability of this ligand to complex
Zr(IV).
The speciation diagrams plotted over the studied pH range (Figure
3) show that ZrL4 species predominate at neutral pH with all ligands.
However, only L1' leads to predominating Zr:L ratios of 1:4 over the
whole studied pH range (from 2 to 12), whereas these ratios
predominate from pH 2 to 9 with L2 and only from pH 5 to 10 with
L3. This suggests that derivatives based on L1' will be useful for 89Zr
biomolecule radiolabeling over a very wide pH range. In addition, it
is noted that from pH 4 to 8, the presence of Zr(OH)4 is significantly
higher for L2 and L3 compared to L1' for which no Zr(IV) hydroxides
were detected below pH 8. This indicates that L2 and L3 are likely to
produce lower radiolabeling yields with 89Zr.
Figure 4. Influence of pH on the % 89Zr complexed after
30 min incubation with 0.4 mM ligand at 20 °C (n ≥ 3)
(+ (▲) and Me-AHA (▼).
with L1' (●), L2 ), L3
Overall, a higher affinity of Zr(IV) for L1’ than for L2 and L3 was
observed. These constants are very close or even higher than those
of Me-AHA that mimics the chelating groups present in DFB. It is
likely that an octadentate chelator based on these HOPO motifs will
lead to very stable Zr(IV) complexes, potentially more stable than
DFB that lacks a fourth coordinating ligand. Indeed, recent work
undertaken by Deri et al. and using the 1,2-HOPO motif supports
this hypothesis.24,34
good agreement with data of the thermodynamic stability study
that also show most favorable results with L1'.
Additional tests were then performed to better probe the kinetic
inertness of the complexes of 1,2-HOPO and Me-AHA against the
presence of excess EDTA or Fe3+. Although the mechanism by which
89Zr is released from its chelate in vivo has not been fully elucidated,
Zr-89 labeling, kinetic stability.
To compare the ability of these ligands to complex Zr(IV) at the
radiotracer level, complexation experiments with 89Zr were
performed. The Me-AHA ligand (Scheme 1) previously investigated
for 89Zr complexation was also included in this study as
reference.18 89Zr]-zirconium(IV) oxalate was incubated under
a
[
various conditions in the presence of a large excess of ligand and
the formation of complexed 89Zr was analyzed using ITLC-SG
chromatographic strips and a 10 mM EDTA solution at pH 6 as
mobile phase. These chromatographic conditions were found to be
the best to distinguish free 89Zr from complexed 89Zr. However,
because of the low kinetic inertness of complexes in the absence of
pre-organization of the "naked" ligands, dissociation occurred with
the weakest ligands during the ITLC-SG elution by transchelation
with the strong competitor EDTA present in the eluent. Whereas
this chromatographic method exhibits limitations to assess the
actual formation of the weakest complexes, it provided essential
information in terms of kinetic inertness, a crucial parameter when
assaying in vivo stability of complexes (see details in ESI).45
Accordingly, the formation of 89Zr complexes was studied from pH 4
to 10 (Figure 4). Results show a drastic superiority of L1' with 89Zr
fully complexed from pH 4 to 8, with a decrease starting only from
pH 9. In contrast, all other ligands tested were significantly less
efficient in retaining 89Zr activity in these conditions, with a
maximum of 2.5 and 3.5 % with L2 and L3, respectively. Whereas far
from L1' efficiency, the reference Me-AHA was superior to L2 and L3
with up to 10% 89Zr complexed visible at pH 8. These results are in
Figure 5. Kinetic inertness of the 89Zr complexes of L1'
and Me-AHA in the presence of a) 50 mM EDTA at pH = 7
at 20 °C (corresponds to 2000 eq. of EDTA over the
ligand) and b) 1mM FeCl3 in phosphate buffer 0.1 M pH
7.2 at 20°C (40 equiv. over the ligand) (n ≥ 3).
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Table 3. Calculated ΔG and ΔH for the Zr(L)4 complexes
formation at 298.15K in the reaction field of water.
Ligand
L1’
L2
L3
Me-AHA
-56.1
-68.9
ΔG (kcal/mol)
ΔH (kcal/mol)
log Ka(exp)
-80.8
-99.8
5.01
-56.8
-76.8
8.83
-58.5
-67.7
9.46
8.75
it is reasonable to invoke transchelation by endogenous chelators
and/or transmetalation by oxophilic endogenous metal cations such
Scheme 3. Chemical structure of Zr-C7 and Zr-L7.20
as Fe3+
.
Moreover, 89Zr complexes that have previously
demonstrated higher inertness than DFB in such in vitro conditions
have often led to improved in vivo stabilities, indicating that such a
simple test can help probing ability of ligand to remain complexed
to the metal in vivo.26,34 L2 and L3 were not considered in this test
since their initial 89Zr complex stability were too weak to provide
accurate and meaningful results. As expected, release of 89Zr in
EDTA was much faster for Me-AHA than L1' with a half-life of the
89Zr-Me-AHA complex < 30 s compared to 5 min for the 89Zr-L1'
complex (Figure 5a). Similarly, the presence of Fe3+ led to a faster
release of 89Zr in the case of Me-AHA (Figure 5b). Overall, in
addition to its better thermodynamic stability reported in the
previous section, the higher inertness of Zr(L1')4 observed at the
radiotracer scale strongly supports the potential superiority of
chelators based on the L1' motif in comparison with hydroxamate
based analogues.
Zr(OH)4 + 4 LH ꢀ Zr(L)4 + 4 H2O (1)
As shown in Table 3, the calculated ΔG for the formation of Zr(L1’)4,
-80.8 kcal/mol, is much more favorable than for Zr(L2)4, Zr(L3)4 and
Zr(Me-AHA)4. When comparing these calculated ΔGs with the
experimental log Ka values, a correlation between the two appears
to exist. Since each log Ka unit corresponds to ΔG of 1.36 kcal/mol,
the 4 log Ka values difference between L1’ and L2 alone may confer
an extra stability of 21.7 kcal/mol (4x4x1.36 kcal/mol) to Zr(L1’)4
over Zr(L2)4 (ΔG = -56.8 kcal/mol). This in turn indicates that the log
Ka of HOPOs is a key determinant in modulating the binding affinity
of HOPOs to Zr(IV). Such log Ka tuning can be achieved by inducing a
H-bond. For example, the calculated log Ka of L1 (that is able to form
intramolecular H-bond via the amide hydrogen) is 1.3 unit lower
than B (in which the possibility of H-bond formation is removed by
replacement of the amide hydrogen by a methyl, see structures in
table 4); such H-bond was observed in the crystal structure of
Zr(L1)4 (see Fig.1B). This log Ka difference between L1 and B
translates to 7.1 kcal/mol in terms of ΔΔG, and thus largely
accounts for the calculated stability of 10.3 kcal/mol for Zr(L1)4 over
Zr(B)4. Whereas the calculated ΔG = -80.8 kcal/mol for Zr(L1')4 is
more negative than the reported cyclic Zr-C7 (ΔG = -71.0 kcal/mol)
and acyclic Zr-L7 (ΔG = -71.0 kcal/mol) tetrahydroxamate complexes
(Scheme 3),20 it is not as kinetically inert against EDTA. Our
previous study has shown that both Zr-C7 and Zr-L7 remain largely
intact (> 80 %) against EDTA even after 7 days while the current
work shows that only 22 % of Zr(L1')4 remain intact against EDTA
after 15 min. The strong Zr-C7 stability thus indicates that the
energy barrier for the dissociation of Zr-C7 must be substantially
higher than that of Zr(L1')4, which highlights the strong impact of the
pre-organization on kinetic inertness of such complexes. This in turn
suggests that L1, L2, and L3 might form complexes of Zr(IV) which can
become as inert or even more inert than the highly stable Zr-C7 if
suitably pre-organized. Of the three, the ligand of choice for further
modification to achieve desirable optimal kinetic inertness appears
to be of the L1 type.
Quantum chemical calculations
To rationalize the thermochemical stability and kinetic inertness
observed experimentally with these complexes, we have resorted
to quantum chemical calculations.
Both ΔG and ΔH for the complexation reaction of Zr(OH)4 with
hydroxypyridinones L were calculated using equation (1) in the
reaction field of water. While the most acidic ligand L1 (or L1') with
a log Ka of 5.01 is deprotonated at neutral pH (unlike L2 and L3, log
Ka of 8.83 and 9.55, respectively), the present calculations were all
done in the neutral form for direct comparison.
Table 4. Calculated impact of the modulation of the 1,2-
HOPO ring on the log Ka of ligands and ZrL4 complex
stability ΔG and ΔH in the reaction field of water at
298.15 K (calculated structure of complexes available in
Figures S5, S6 and S7).
A
B
L1
log Ka(calc)
5.8
5.8
4.5
ΔGZrL4(kcal/mol)
ΔHZrL4(kcal/mol)
-72.9
-82.3
-78.1
-91.7
-88.4
-99.0
Comparison of HOPOs with hydroxamates
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Most data reported herein can be compared to results reported However, it must be kept in mind that the 3,2-HOPO and 3,4-
previously for hydroxamate ligands.18,20 Both ligand classes exhibit a HOPO rings we reported herein have the advantage of
strong predominancy of Zr(IV) octacoordination by four ligands at providing ZrL4 complexes of higher aqueous solubility. Thus, by
physiological pH, suggesting that chelators made of 4 of these tuning them to provide lower pKa and/or generate H-bond and
ligands are likely to provide optimal stability for in vivo use. The amide-π interactions as in 1,2-HOPO, they can also become
thermodynamic stability study did not highlight noticeable highly adequate for the design of Zr(IV) chelators. We believe
differences between the HOPOs L2 and L3 and the hydroxamate that the crystallographic, thermochemical and quantum
ligand (pZr4+ in the 28.5-28.6 range), whereas L1' was significantly chemical studies presented herein will provide a firm basis for
superior (pZr4+ = 33.48). Differences appear more marked when such task and for further advances in the growing field of PET
comparing the inertness of complexes. At radiotracer imaging with 89Zr.
concentrations, L1' exhibited by far the highest inertness in
presence of competitive ligand or metal, followed by Me-AHA that
was in turn significantly superior to L2 and L3. Such results provide
good indications of the potential of these ligands for
radiopharmaceutical applications with 89Zr. Despite the lower
inertness of Me-AHA complexes than L1', hydroxamates offer the
advantage of smaller and simpler structures. This may be a criterion
of choice for designing chelators. They benefit of being already used
in clinical trials for several years with 89Zr with the FDA approved
DFB ligand, which might facilitate the clinical transfer of future new
chelators based on that same pattern. Recent works undertaken in
several research group highlight the interest of working with the
Experimental
General. Procedures for ligands preparation, crystal structure
analysis, thermodynamic stability constants determination and
complexation at the radiotracer level with 89Zr are available in the
Supplementary Information file.
Preparation of natZrL4 complexes
Zr(L1)4 To zirconium(IV) acetylacetonate (acac) (102 mg, 205
.
µmol) in dry DMF (10 mL) was added L1H (155 mg, 927 µmol)
dissolved in dry MeOH (10 mL) and the solution was refluxed
for 18 h. After cooling down to room temperature, the white
precipitate was filtered and dried in vacuo (85 mg, 55%). 1H
NMR (DMSO-d6, 300 MHz, ppm): δ 2.94 (d, 3H), 7.04 (d, 1H, J =
8.7 Hz), 7.34 (d, 1H, J = 7.5 Hz), 7.67 (m, 1H), 9.57 (s, 1H
broad). 13C NMR (DMSO-d6, 75 MHz, ppm): δ 26.2, 113.3,
115.5, 135.8, 137.1, 159.1, 160.3. ESI-MS: m/z = 781.1
[M+Na]+, 675.1 [M-L1+DMSO-d6]+ (+ Zr isotopic distribution,
see spectrum in ESI). Elemental analyzes: Calculated for
C28H28N8O12Zr + 4 H2O: C, 40.43; H, 4.36; N, 13.47%. Found: C,
40.23; H, 4.42; N, 13.09. Crystals suitable form X-ray diffraction
were obtained by recrystallization in hot acetonitrile (Details in
ESI).
hydroxamate
motif.25,26
On
the
other
hand,
the
hydroxypyridinonate structure offers the advantage of a higher
modularity since the hydroxyl group or the substituents position
can be varied, offering more options to tune the characteristics of
Zr(IV) chelators. This is reflected in the variety of HOPO isomers
used in chelators that have been reported recently30,32,34 and we
hope results presented herein will guide future developments in
that field.
Conclusions
Recent literature shows a strong interest in improving the
complexation of 89Zr for PET imaging applications. From only 1
available and suboptimal ligand (desferioxamine B) until 2013,
more than 10 chelators have now been developed by several
research groups to provide enhanced in vivo stability of the
radiolabeling. Whereas several HOPO-based chelators have
been proposed, with very promising results for some of them,
the literature strongly lacks data on the basics of the
complexation of Zr(IV) by this class of bidentate ligands. Yet,
such data is essential to finely tune the characteristics of
chelators to match the coordination sphere of the Zr(IV)
cation. In this study, which compares three hydroxypyridinone
positional isomers, we observed that the selected 1,2-HOPO
pattern combines the best features to provide high
thermodynamic stability and high kinetic inertness for Zr(IV)
complexation. The origin of this superiority arises in part from
its higher acidity, but also from H-bonding and amide-π
interactions that were not observed in the 2,3-HOPO and 3,4-
HOPO used in this study. The 1,2-HOPO isomer, in combination
with a H-donor amide group next to the hydroxyl, appears as a
powerful pattern to build octadentate chelators for Zr(IV)
complexation. These observations are in line with the
promising results reported recently by Deri et al with a similar
1,2-HOPO based chelator that showed high in vivo inertness.34
Zr(L1')4. Was prepared from L1'H and Zr(acac)4 using the same
procedure as Zr(L
1)4, except that the product did not
precipitate upon formation. After evaporation of methanol,
the residue was dissolved in chloroform and washed twice
with water. The chloroform was evaporated, affording a
1
yellowish solid (97 %). H NMR (CDCl3, 300 MHz, ppm): δ 1.02
(t, 3H, J = 7.2 Hz), 1.64 (m, 2H), 3.37 (m, 2H), 6.90 (d, 1H, J =
8.4 Hz), 7.51 (m, 1H), 7.61 (m, 1H) 9.95 (t, broad, 1H). 13C NMR
(CDCl3, 75 MHz, ppm): δ 11.8, 22.7, 42.0, 114.7, 116.2, 135.2,
137.1, 158.8, 161.1. ESI-MS: m/z = 870.9 [M+H]+, 892.9
[M+Na]+ (+ Zr isotopic distribution, see spectrum in ESI).
Elemental analyzes: Calculated for C36H44N8O12Zr + 6 H2O: C,
44.12; H, 5.76; N, 11.43%. Found: C, 43.96; H, 5.88; N, 11.80.
Zr(L2)4. Was prepared from L2H and Zr(acac)4 using the same
procedure as Zr(L1)4, except that the product did not
precipitate upon formation. After evaporation of methanol,
the residue was partitioned between water and chloroform.
The water was evaporated, affording a white solid (88 %). 1H
NMR (DMSO, 300 MHz, ppm): δ 2.15 (t, 2H, J = 7.2 Hz), 2.55 (d,
3H), 3.90 (t, 2H, J = 7.2 Hz), 6.35 (d, 1H), 6.88 (m, 1H), 7.58 (d,
1H). 13C NMR (DMSO-d6, 75 MHz, ppm): δ 25.4, 33.7, 45.8,
8 | J. Name., 2012, 00, 1-3
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110.8, 113.3, 122.2, 156.9, 163.0, 169.5. ESI-MS: m/z = 675.1
[M-L2]+, 757.1 [M-L2+2MeOH+H2O]+, 897.1 [M+H2O+H]+ (+ Zr
isotopic distribution, see spectrum in ESI). Elemental analyses:
Calculated for C36H44N8O12Zr + 6 H2O: C, 42.12; H, 5.76; N,
11.43%. Found: C, 44.22; H, 5.42; N, 11.51. Crystals suitable for
X-ray diffraction were obtained by slow evaporation from a
50:50 mixture of ethyl acetate and methanol (Details in ESI).
Zr(L3)4. Was prepared from L3H and Zr(acac)4 using the same
procedure as Zr(L1)4, except that the product did not
precipitate upon formation. The reaction solution was reduced
to about 2 mL under reduced pressure and the product
precipitated as a yellowish solid upon addition of Et2O. The
solid was filtered and dried in vacuo (69%). 1H NMR (DMSO,
300 MHz, ppm): δ 2.40 (s, 3H), 2.54 (d, 3H), 2.63 (t, 2H, J = 6.6
Hz), 4.38 (t, 2H), 6.49 (m, broad, 1H), 7.77 (m, broad, 1H), 8.04
(m, broad, 1H). 13C NMR (DMSO-d6, 75 MHz, ppm): δ 11.1,
25.4, 36.1, 50.0, 106.0, 126.6, 133.0, 133.3, 158.6, 169.1. ESI-
MS: m/z = 717.2 [M-L3]+ (+ Zr isotopic distribution, see
spectrum in ESI). Calculated for C40H52N8O12Zr + 6 H2O: C,
46.36; H, 6.23; N, 10.81%. Found: C, 46.02; H, 5.87; N, 10.57.
Notes and references
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Crystal structure coordinates of Zr(L1
)
were used as a
4
template to build Zr(
structures and the crystal structure coordinates of Zr(
Zr(L2
were then geometry optimized by utilizing the M06L
L
1')4, Zr(
L
3)4, Zr(
A
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and Zr(
B
)4. These
1)4 and
4
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Acknowledgements
This research was supported by the Intramural Research
Program of the NIH, National Cancer Institute, Center for
Cancer Research and Center for Information Technology and in
part by grants from the French National Agency for Research,
called “Investissements d’Avenir” IRON Labex n° ANR-11-LABX-
0018-01 and ArronaxPlus Equipex n°ANR-11-EQPX-0004 and
the Cancéropôle Grand Ouest. The quantum chemical study
utilized PC/linux clusters at the center for Molecular Modeling
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center, for providing 89Zr.
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Hydroxypyridinones were studied in their most simple form for the complexation of Zr(IV) which
provides important data for the design of pre-organized chelators of this metal for nuclear imaging
applications.