Evaluation of the Tetrakis(3-Hydroxy-4-Pyridinone) Ligand THPN with Zirconium(IV): Thermodynamic Solution Studies, Bifunctionalization, and in Vivo Assessment of Macromolecular89Zr-THPN-Conjugates

Article abstract of DOI:10.1021/acs.inorgchem.9b02350

Zirconium-89 (⁸⁹Zr) is a suitable radionuclide for positron-emission tomography (PET) of long-circulating targeting vectors such as monoclonal antibodies (mAbs). Due to stability concerns for the most widely used ⁸⁹Zr-chelating agent

Full text of DOI:10.1021/acs.inorgchem.9b02350

Article
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/IC
Evaluation of the Tetrakis(3-Hydroxy-4-Pyridinone) Ligand THPN
with Zirconium(IV): Thermodynamic Solution Studies,
89
Bifunctionalization, and in Vivo Assessment of Macromolecular Zr-
THPN-Conjugates
,
†
z,‡ Julie Rousseau,
§
Christian Buchwalder,* María de Guadalupe Jaraquemada-Pelae
́
§
†,∥
‡
ard, Paul Schaffer,⊥
§
Helen Merkens, Cristina Rodríguez-Rodríguez,
̈
Katayoun Saatchi,* and Urs O. Hafeli*
Chris Orvig, Franco
̧
is Ben
́
,
†
,†
†
University of British Columbia, Faculty of Pharmaceutical Sciences, 2405 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada
Medicinal Inorganic Chemistry Group, University of British Columbia, Department of Chemistry, 2036 Main Mall, Vancouver, BC
‡
V6T 1Z1, Canada
§
BC Cancer Agency, Department of Functional Imaging, 600 West 10th Avenue, Vancouver, BC V5Z 4E6, Canada
∥
University of British Columbia, Department of Physics & Astronomy, 6224 Agricultural Road, Vancouver, BC V6T 1Z1, Canada
⊥
TRIUMF, Life Sciences Division, 4004 Wesbrook Mall, Vancouver, BC V6T 2A3, Canada
S Supporting Information
*
89
ABSTRACT: Zirconium-89 ( Zr) is a suitable radionuclide
for positron-emission tomography (PET) of long-circulating
targeting vectors such as monoclonal antibodies (mAbs). Due
to stability concerns for the most widely used 89Zr-chelating
agent desferrioxamine B (DFO) in preclinical studies,
8
9
alternative Zr-chelators are currently being developed. We
recently reported on the first tetrakis(3-hydroxy-4-pyridinone)
(
3,4-HOPO) ligand THPN, which was identified as a
8
9
promising Zr-chelator. In this study, we aimed to further
explore this octadentate chelate in vitro and in vivo. The
IV
[
Zr (THPN)] thermodynamic stability was quantified in solution titration studies, which revealed one of the highest formation
constants reported for a zirconium chelate (log βML 50.3(1), pM = 42.8). Solution stabilities with iron(III) were also
III
exceptionally high and can compete with some of the strongest Fe -chelates. A first bifunctional derivative of the octadentate
8
9
ligand, p-SCN-Bn-THPN, was then produced in a multistep synthesis. To assess and compare the long-term Zr complex
stability, bifunctional THPN, as well as the literature chelators p-SCN-Phe-DFO and p-SCN-Phe-DFO*, werre conjugated to
the high-molecular weight (800 kDa) polymeric carrier hyperbranched polyglycerol (HPG). The functionalized HPGs were
radiolabeled with 89Zr , and the integrity of the radioconjugates was assessed over several days in vitro and in vivo. While all
three radioconjugates remained >95% intact over 5 days in human plasma, the in vivo study in healthy mice revealed higher
physiologic stability of the DFO and DFO* radiochelates over bifunctional THPN conjugates. This was evidenced by increased
IV
89
IV
bone uptake of osteophilic Zr for THPN. This finding contrasts with the exceptionally high thermodynamic stability of the
chelate and suggests either a kinetic or metabolic lability, or may stem from coordinative changes due to the covalent
conjugation of the 89Zr-THPN radiochelate as suggested by density functional theory (DFT) calculations. These important
findings inform the design of next generation 3,4-HOPO chelates with the aim of improving the physiologic stability. This study
furthermore demonstrates how HPG can be used as a robust carrier tool to assess and compare the long-term in vivo stability of
radiochelates.
INTRODUCTION
targeting vectors such as mAbs. Currently the most widely used
Zr-chelator is the bacterial siderophore desferrioxamine B
■
8
9
Zirconium-89 ( Zr, t = 78.41 h) is a suitable radionuclide for
1/2
(
DFO), which is used in the majority of clinical trials
positron-emission tomography (PET) of long-circulating
targeting vectors. As such, it has gained much attention for
immunoPET imaging of monoclonal antibodies (mAbs), which
possess a relatively slow pharmacokinetic biodistribution
8
9
investigating Zr-imaging agents. However, rodent data have
led to concerns over the in vivo stability of Zr-DFO complexes,
8
9
1
,2
profile. Most commonly, bifunctional chelators (BFCs) are
Received: August 2, 2019
employed to coordinate the radiometal ion and tether it to
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.9b02350
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Inorganic Chemistry
Article
Figure 1. Selection of bifunctional 89Zr-chelators discussed in this work. Coordinating oxygen atoms are highlighted in bold.
which has recently motivated the development of alternative
chelating agents. A number of different chelators have been
the evolution of spectral features can be found in the Supporting
3
,4
Information. Analysis of the combined spectrophotometric−
33
investigated with variable success, and recent progress has been
potentiometric data with the software HypSpec2014 allowed
us to determine all ten protonation constants of THPN (Table
1) and the generation of the corresponding speciation plot
5
−9
the topic of several reviews. Several groups modified DFO by
expanding its hexadentate denticity by addition of two donor
1
0−13
groups,
ligand (Figure 1).
chelator scaffolds based on hydroxamic acid,
such as in the design of the octadentate DFO*
(
Figure 2). It shows that at a physiologic pH of 7.4, THPN exists
14,15
Others explored entirely different
predominantly in its neutral form H THPN.
4
1
6−22
acetic
2
3
24
25
acid, picolinic acid, hydroxyisophthalamide, 2,3-
dihydroxyterephthalamide (TAM), or hydroxypyridinone
a
Table 1. Protonation Constants of THPN
2
6
2
7−32
89
equilibrium reaction
log β
log K
(
HOPO) groups
Our laboratory recently reported on the first chelator based on
four 3-hydroxy-4-pyridinone (3,4-HOPO) groups, named
as alternative Zr-chelators.
+
L + H ⇆ HL
10.28(2)
20.11(2)
29.41(2)
38.12(2)
42.43(3)
46.14(3)
49.35(2)
52.49(2)
54.97(1)
56.77(2)
10.28(2)
9.83(2)
9.30(2)
8.71(2)
4.31(3)
3.71(3)
3.21(2)
3.14(2)
2.48(1)
1.80(2)
+
HL + H ⇆ H
L + H ⇆ H
L + H ⇆ H
L + H ⇆ H
L + H ⇆ H
H L + H ⇆ H L
H L + H ⇆ H L
H L + H ⇆ H L
L
2
2
7
+
THPN. This octadentate ligand was found to be a suitable
H
H
H
H
2
3
4
5
3
4
5
6
L
8
9
+
Zr-chelator that showed fast radiolabeling kinetics, promising
L
L
L
+
in vitro chelate stability and favorable pharmacokinetic clearance
in vivo. Encouraged by these findings, we set out to further
investigate the stability of this chelate system in vitro and in vivo.
Thermodynamic complex formation constants of THPN were
+
+
6
7
+
7
8
IV
III
+
determined with Zr and Fe in spectrophotometric and
potentiometric solution studies. We then produced a bifunc-
tional derivative, p-SCN-Bn-THPN (Figure 1), in a multistep
synthesis to allow for covalent conjugation to carriers. To assess
long-term in vivo radiocomplex stability, p-SCN-Bn-THPN
along with bifunctional DFO and DFO* derivatives were
conjugated to hyperbranched polyglycerol (HPG) particles and
8
9
+
H L + H ⇆ H L
9
10
a
Determined by simultaneous UV-potentiometric titrations at 25 °C,
I = 0.16 M NaCl. Charges are omitted for clarity.
8
9
IV
radiolabeled with Zr , and the stability of the three resulting
radioconjugates was assessed over several days in vitro, in
plasma, and in vivo, by PET/CT imaging and biodistribution
studies.
RESULTS AND DISCUSSION
■
Thermodynamic Solution Studies. To be viable radio-
pharmaceuticals, radiometal chelates need to provide high
thermodynamic stability and kinetic inertness. In order to
quantify the thermodynamic stability of the Zr-THPN complex,
we investigated the THPN ligand and its Zr - and Fe -complex
formation equilibria in several solution studies.
IV
III
IV
THPN Ligand Protonation Constants. Since the Zr -affinity
of the THPN ligand, and in particular of its HOPO groups,
depends on the acid−base properties, the protonation equilibria
of the THPN ligand were first determined. Spectrophotomet-
ric−potentiometric titrations of an acidic aqueous THPN
solution were carried out in the pH range 1.7−10.5 by addition
of standardized NaOH and were measured at 25 °C and with an
ionic strength (I) of 0.16 M NaCl. A more detailed discussion of
Figure 2. Speciation plot for the THPN ligand calculated with
protonation constants in Table 1, [THPN] = 8.47 × 10 M; the
dashed line represents pH = 7.4.
−
5
B
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Inorganic Chemistry
Article
Complex Formation Equilibria with FeIII and Zr . The
IV
and [Fe(HTHPN)] complexes upon addition of Zr4+
equivalents (Figure 4 and Figure S3). In addition, we observed
a slight red shift of the UV band centered at 290 to 295 nm upon
Zr -transmetalation of the Fe -THPN complexes (Figure 4 left
inset, Figure S3, and Figure S4). The pH was held constant at
exceptional strength of the metal complexes formed between
THPN and Zr required a combination of different methods to
determine the Zr -THPN stability constants. Thus, the stability
constants of a Fe -THPN system were first determined,
followed by a Fe −Zr metal−metal competition experiment.
From combining these results, the Zr -THPN stability
IV
IV
IV
III
III
III
IV
pH 2 to avoid precipitation of iron hydroxides.
IV
2+
The stability constants of the [Zr(H THPN)] species could
2
III
constants could then be determined.
then be calculated from comparison with the spectra of the Fe -
III
The Fe -THPN formation constants were determined by
THPN complexes present at pH 2 and are included in Table 2.
In additional direct UV-potentiometric titrations we found that
direct UV−vis batch proton competitions, as well as by direct
2
+
simultaneous potentiometric−spectrophotometric titrations.
[Zr(H THPN)] deprotonates its two protons with dissocia-
2
III
The formation of the Fe -THPN complexes is observed by
tion constants of pK = 3.3 and 3.9, respectively, to yield the
a
spectroscopic evolutions of the system as the pH was raised in a
batch titration experiment (Figure S2). From pH 0.16−1.47, a
new band emerges centered at 510 nm, which is characteristic of
neutral [Zr(THPN)] complex. These deprotonations are
accompanied by a shift of the band at 295 to 305 nm and the
occurrence of two isosbestic points at 296 and 295 nm (Figure
S4). At pH ∼ 6.5 the neutral [Zr(THPN)] complex started to
precipitate and the potentiometric data were therefore not
included in the calculations. However, as shown by the UV
spectra in Figure S4, the [Zr(THPN)] complex does not exhibit
further transformations and remains stable at higher pH. The
III
red colored Fe -HOPO complexes and indicates the formation
III
+
of the [Fe (H THPN)] complex from the colorless
2
III
3+
[
Fe (OH ) ] in solution. In the pH range 1.47−3.26, a new
2
6
band appears at 458 nm and two isosbestic points are observed
at 360 and 503 nm, suggesting two species in equilibrium. This is
attributed to the deprotonation of a hydroxy group from one
IV
distribution diagrams for the Zr -THPN complexes were
calculated using the stability constants presented in Table 2
and are shown in Figure 3b.
HOPO unit (pK = 2.01), which leads to the formation of the
a
III
III
fully coordinated Fe -complex [Fe (HTHPN)]. Finally, the
last hydroxy substituent deprotonates with pK = 8.23 to form
The stability constants of the [Zr(THPN)] and the
a
the negatively charged [Fe(THPN)]− species. The UV−vis
spectrum remains unchanged for this last deprotonation,
indicating that the last HOPO unit is not involved in the
−
[Fe(THPN)] complexes are both exceptionally high (log
β
= 50.3(1) and log βML = 44.71(7), respectively), with the
ML
IV
III
Zr complex stability exceeding that of the Fe complex. This
III
coordination sphere. The Fe -complex formation by THPN is
confirms that THPN is capable of forming a thermodynamically
IV
further confirmed by a red shift of the free protonated ligand
stable Zr -complex. To the best of our knowledge, the stability
from 282 to 290 nm as the pH is raised from 0.16 to 3.26. The
constant for the [Zr(THPN)] complex is the highest reported
III
IV
spectroscopic features in the formation of the Fe -THPN
stability constant for a Zr complex in aqueous solution thus far.
complexes evolve similarly to those of the clinically used iron
chelator deferiprone and other bidentate deferiprone analogs in
the formation of their 1:1, 1:2, and 1:3 metal-to-ligand
The closest reported value is for the [Zr(3,4,3-LI(1,2-HOPO))]
complex and is 7 orders of magnitude lower (log β
=
ML
37
43.1(6)). The stability constants for the Zr-DFO system have
3
4−36
complexes.
Table 2 presents the stability constants
only recently been investigated by two groups that obtained
38
different results. Bianchi and co-workers reported the Zr-DFO
Table 2. Stability Constants for the Complexes Formed by
system to predominantly form polynuclear M L and M L
2
2
2 3
3
+
4+
THPN with Fe and Zr Ions at 25 °C and with I = 0.16 M
species, besides the 1:1 complex. Toporivska and Gumienna-
39
NaCl
Kontecka, on the other hand, reported the presence of only the
monometallic species, for which they determined a log βMHL of
III
IV
Fe -THPN
Zr -THPN
4
7.6(3). Comparing this reported Zr-DFO species with
model
log β
pKa
log β
pKa
+
protonated [Zr(HTHPN)] (log β
54.2(1)), the THPN
MHL
a
a
b
c
MH2L
MHL
ML
54.95(1)
52.94(1)
44.71(7)
38.0
57.51(5)
complex is 6.6 orders of magnitude stronger than the DFO
complex.
While the stability constant is a good measure for the overall
thermodynamic complex stability in solution, an even more
effective way to compare different chelators for biological
applications is by employing pM values. pM is linearly correlated
b
2.01
8.23
54.2(1)
3.3
3.9
b
50.3(1)
pM
42.8
a
b
Determined by proton competition UV−vis titrations. Determined
c
by simultaneous UV-potentiometric titrations. Determined by
metal−metal competition UV−vis titrations. Charges are omitted
for simplicity.
to the stability constant of the metal complexes at physiolog-
n+
ically relevant conditions and is defined as pM = −log [M ]
free
n+
x−
40,41
at pH 7.4 with [M ] = 1 μM and [L ] = 10 μM.
Besides the
III
determined for the Fe -THPN complexes by refinement of
thermodynamic stability constants of the metal complexes, the
pM value also takes ligand acid−base properties into account,
allowing for the most suitable comparison of the ability of
3
3
the spectroscopic data with the HypSpec2014 software. The
corresponding speciation plot is shown in Figure 3a.
Next, an indirect metal−metal competition UV−vis titration
was performed to determine the stability constants of the Zr -
different ligands with diverse basicities and/or denticities and
IV
42,43
different metal-to-ligand stoichiometries.
Even though the
THPN complexes, as the stability constants were too high to be
determined by direct methods. This method exploited the fact
[Zr(THPN)] complex has the higher overall stability constant,
the higher ligand basicity of THPN results in a 1.2 unit lower pM
value for [Zr(THPN)] when compared to [Zr(3,4,3-LI(1,2-
HOPO))] (Table 3). Still, both HOPO complexes exert
impressively high thermodynamic stability. The pM value for
III
IV
that, other than the Fe -THPN complexes, the Zr -THPN
complexes are spectroscopically silent in the visible range. The
III
IV
competition between the Fe - and the Zr -complexes could
IV
therefore be observed by the disappearance of the red ligand-to-
the monometallic DFO complex with Zr was determined as
+
39
metal charge transfer (LMCT) band of the [Fe(H THPN)]
32.2, which is 10 orders of magnitude lower than that for
2
C
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Inorganic Chemistry
Article
III
IV
Figure 3. Speciation plots for (a) Fe -THPN complexes and (b) Zr -THPN complexes calculated with stability constants from Table 2. [THPN] =
[
n+
−4
M ] = 1.10 × 10 M; dashed line indicates pH = 7.4.
III
4+
3+
−5
4+
Figure 4. Metal−metal competition spectra of the Fe -THPN system with increasing equivalents of Zr . [THPN] = [Fe ] = 7.16 × 10 M; [Zr ]/
3
+
4+
[
Fe ] = 0−4.5; pH = 2, 25 °C, I = 0.16 M NaCl, l = 1 cm. The insets represent the change in absorbance with increasing equivalents of Zr at λ = 279,
00, or 305 nm (left) and at λ = 427, 475, or 510 nm (right), respectively.
3
IV
Table 3. Comparison of pM Values for Zr -Complexes with
THPN or Literature Chelators
treatment of iron overload disease or as antimicrobial agent by
iron deprivation.
Bifunctional Ligand Design and Synthesis. In order to
conjugate the THPN chelator to carrier or targeting molecules, a
bifunctional derivative, p-SCN-Bn-THPN, was produced in a
nine-step synthesis (Scheme 1). As a bifunctional handle, we
opted to install a para-isothiocyanatobenzyl group at the central
carbon of the propylene-bridged THPN backbone. Positioned
in this way, the bifunctional linker would point away from the
metal center (as supported by density-functional theory (DFT)
simulations, cf. Figure S39), thereby facilitating conjugation,
while at the same time preventing the introduction of a center of
chirality. The bifunctional handle was introduced at the
beginning of the nine-step synthesis in the form of a protected
p-nitrobenzyl group. The single alkylation of diethyl malonate
with 4-nitrobenzyl bromide was found to work best using
sodium hydride as the base in tetrahydrofuran as previously
THPN 3,4,3-LI(1,2-HOPO)
DTPA
DFO
citrate
a
a
,
b
c
,
ab
pM
42.8
44.0
∼33.9
c
32.2
∼28.7
a
b
From ref 37. Calculated, from ref 44. From ref 39.
THPN. pM values based on computational approximations have
also been reported for the Zr-complexes of DTPA and
3
7,44
citrate,
and the values are included in the comparison in
Table 3. Hence, from a thermodynamic stability point of view,
both THPN and 3,4,3-LI(1,2-HOPO) appear to be much more
effective in sequestering Zr4 and form much stronger Zr -
+
IV
44
complexes than DFO, DTPA, or citrate.
These results further indicate that THPN is also an excellent
III
III
Fe -chelator. The pM value for Fe -THPN (pM = 38.0)
exceeds that of the iron(III) chelate with the siderophore
47
48
reported. Attempts to reproduce a literature procedure that
uses sodium ethoxide in ethanol produced, in our hands, the
undesired dialkylated product. The functionalized backbone
45,46
enterobactin (pM = 34.3),
one of the strongest iron
chelators. THPN may therefore hold therapeutic potential for
D
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Inorganic Chemistry
Article
a
Scheme 1. Synthesis of the Bifunctional Chelator p-SCN-Bn-THPN
a
Reagents and conditions: (i) NaH, THF, 35 °C, 53%; (ii) NH , MeOH, RT, 89%; (iii) BH ·THF, THF, RF, 66%; (iv) Na CO , DMF, RT, 45%;
3
3
2
3
(
v) H PO , MeCN, 45 °C, 97%; (vi) DCC, HOBt, DMF, MW 55 °C, 83%; (vii) AcOH/HCl (conc.), 50 °C, 94%; (viii) H , Pd/C, MeOH, RT,
3
4
2
9
9%; and (ix) CSCl , CHCl /H O, RT, 65%.
2 3 2
synthon was then converted into the diamine 3 by amination,
followed by reduction with borane. Next, the four pendant arms
were introduced by alkylation of the amines with tert-butyl
bromoacetate, followed by ester hydrolysis using aqueous ortho-
phosphoric acid to give the tetraacid 5. Using DCC/HOBt-
mediated amide coupling, four equivalents of benzyl-protected
HOPO-methylamine 6 (2-(aminomethyl)-1,6-dimethyl-3-
building block 6 was purposefully introduced at a later stage, and
the cumulative yield of ∼50% over the remaining four reaction
steps was reasonably high.
89Zr-Chelate-Hyperbranched Polyglycerol (HPG) Ra-
dioconjugates. In order to evaluate the long-term stability of
89
the Zr-THPN radiocomplex and compare the ligand with the
hydroxamic acid-based literature chelators DFO and DFO*,
high-molecular weight (∼800 kDa) hyperbranched polyglycerol
(
phenylmethoxy)-4(1H)-pyridinone) were then conjugated to
the tetraacid to give the protected precursor 7. Deprotection of
the HOPO groups and the bifunctional handle, followed by
activation of the latter, was then performed in three separate
steps to allow best reaction control. This produced the activated
bifunctional ligand p-SCN-Bn-THPN (10) in a cumulative yield
of ∼7% (nine steps, Scheme 1). It is worth mentioning that most
of the lower-yielding reactions occurred at the beginning of the
multistep synthesis where starting materials were readily
available and relatively inexpensive. The more costly HOPO-
(
HPG) was selected as carrier. HPG particles are highly
biocompatible and can be readily synthesized as globular
49−55
macromolecules with narrow size distributions.
They have
been investigated for a number of applications including as drug
5
6
57
delivery vehicles, blood plasma expanders, cell surface
58
54,55,59
protectants, or carriers for diagnostic imaging agents.
We reasoned that the long circulation half-life of high-molecular
weight HPG would render these polymeric macromolecules also
E
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Table 4. In Vitro Plasma Stability of 89Zr-Chelate-HPG
Radioconjugates
suitable carriers to investigate the long-term in vivo stability of
attached radiochelates.
a
The three bifunctional chelators p-SCN-Bn-THPN, p-SCN-
Phe-DFO, and p-SCN-Phe-DFO* (Figure 1) were thus
radioconjugate
initial
1 d
3 d
5 d
89Zr-THPN-HPG
89Zr-DFO-HPG
89Zr-DFO*-HPG
>99 ± 0
>99 ± 0
>99 ± 0
97 ± 1
>99 ± 0
>99 ± 0
96 ± 1
>99 ± 0
>99 ± 0
96 ± 1
97 ± 0
conjugated to amine-functionalized HPG (HPG-NH ) of
2
∼
16.7 ± 0.2 nm hydrodynamic diameter (Z-avg.) by thiourea
>99 ± 0
bond formation. After overnight incubation, the modified HPG-
chelator conjugates were purified by size-exclusion chromatog-
raphy (SEC) and ultrafiltration. Analyses by dynamic light
scattering (DLS) indicated that chelator modifications did not
significantly affect particle size and size distribution, which was
further supported by indistinguishable size-exclusion HPLC
aStability over time is reported as the percentage of bound 89Zr after
incubation in human plasma at 37 °C and at 350 rpm. Measurements
were performed in triplicate and are reported as the mean ± SD.
incubation in human plasma. These results encouraged us to
further investigate the radiocomplex stability in a healthy mouse
model.
(
SE-HPLC) chromatograms (Figure 5 a, b).
In Vivo Stability Study. In our previous report on the THPN
8
9
ligand, we studied the in vivo stability and excretion of the Zr-
THPN chelate by itself. Due to the fast clearance of the
radiocomplex, the question for the long-term in vivo stability
could not be investigated. In order to address this long-term in
8
9
vivo stability of Zr-THPN and compare it against DFO and
DFO* radiochelates, we conducted an in vivo study with the
chelates tethered to HPG macromolecules. To this end, healthy
8
9
NSG mice were injected with Zr-chelate-HPG of either of the
three chelates. Each radioconjugate was administered intra-
venously (i.v.) in order to perform biodistribution studies, and
mice were sacrificed after either 1, 3, or 6 days post-injection
(
p.i.) (n = 4 per group and time point). One animal of each
group received a larger dose of radioconjugate and was imaged
by PET/CT after 1 h, 1 d, 3 d, and 6 days p.i., before being
included in the ex vivo biodistribution study.
PET images at 1 h and 1 d p.i. showed the majority of activity
for all three radioconjugates to be associated with the blood
circulation as hearts and carotid arteries were clearly distinguish-
Figure 5. (Radio-)SE-HPLC chromatograms of (a) unconjugated
8
9
HPG-NH , (b) chelator-modified HPG, and (c) Zr-chelate-HPG
2
radioconjugates show no appreciable difference upon conjugation or
radiolabeling with retention times of 15.1−15.3 min.
8
9
able (Figures 6 and S42). After 3 and 6 days p.i., Zr uptake in
8
9
the joints and bones of the mouse that received Zr-THPN-
HPG was noticeably higher than for the other two mice
receiving the DFO and DFO* radioconjugates. All three mice
also showed some activity uptake by liver and spleen.
HPG-chelator solutions of either of the three chelators
(
THPN, DFO, or DFO*) were then radiolabeled at ambient
89
temperature with Zr-oxalate solutions (∼32 MBq, ∼870 μCi).
Once analysis by instant thin-layer chromatography (ITLC)
indicated quantitative radiolabeling, reaction mixtures were
purified by SEC and the radioconjugates were recovered in 97%
radiochemical yield for all three conjugates. The isolated
radioconjugates were analyzed by ITLC and radio-SE-HPLC,
which indicated excellent radiochemical purity (>99%) and
showed matching retention times and peak shapes compared
The biodistribution data confirmed these findings (Figure 7)
and are presented in Table 5 and in Figures S40 and S41.
8
9
3,60,61
Since released Zr accumulates in bones,
bone uptake
8
9
can be regarded as a measure of released Zr and thus of
radiocomplex instability. The in vivo data from PET/CT
imaging and biodistribution studies both suggest that the DFO-
and DFO*-radioconjugates possess superior in vivo stability in
8
9
with HPG-NH and chelator-modified HPGs (Figure 5). DLS
mice over the Zr-THPN-HPG radioconjugate. Based on the
exceptionally high thermodynamic stability measured for the
[Zr(THPN)] complex (log βML = 50.3(1), pM = 42.8, vide
supra), we did not anticipate this finding.
2
analysis further showed that the hydrodynamic diameter and
polydispersity of the particles remained largely constant upon
radiolabeling.
In Vitro Plasma Stability Study. As a first approximation for
physiologic stability of the radioconjugates, an in vitro plasma
stability study was conducted. 89Zr-chelate-HPG particles with
either THPN, DFO, or DFO* as chelators were diluted 4-fold in
human blood plasma and were incubated for 5 days at 37 °C
with agitation. Aliquots were analyzed by ITLC after 1, 3, and 5
days. The fraction of intact radiocomplex was quantified by
Exploring different explanations for this discrepancy, we
conducted DFT geometry optimizations to compare a thiourea-
containing surrogate of the conjugated Zr-THPN complex
(Figure S39) and compared it to the nonbifunctional Zr-THPN
complex by itself (Figure S38). Interestingly, the introduction of
the conjugation group seems to cause a small disturbance of the
coordination geometry. This is evidenced by an elongation of a
Zr−O bond from one ketonic HOPO oxygen from 2.249 to
2.331 Å, while the other Zr−O bond lengths remained largely
unchanged (Table S2). This change in the coordination
geometry may lead to a reduced thermodynamic stability of
the bifunctional complex when compared to the complex itself.
Another possible explanation for the low in vivo stability is the
fact that physiologic stability is not only based on the
89
integration of the bound fraction of Zr on ITLC plates, where
8
9
complexed Zr remained close to the baseline (R ∼ 0), while
f
8
9
released Zr migrated with the mobile phase front (R ∼ 1). The
f
obtained results for the three radioconjugates are shown in
Table 4. The DFO* radioconjugate showed no signs of
dissociation, and the other two radioconjugates remained
largely intact and prevailed with >95% integrity after 5 days of
F
DOI: 10.1021/acs.inorgchem.9b02350
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
metal ions and native chelators for potential transmetalation or
transchelation. These challenges, together with the changes in
the coordination environment upon conjugation, may explain
the unsatisfactory in vivo performance. These insights are
valuable in the design of next-generation 3,4-HOPO-based
chelates. These should be conceptualized to provide higher
kinetic inertness (such as by using a macrocyclic scaffold) and to
find an improved bioconjugation linker that does not affect the
coordination environment.
While different in vitro methods exist to study the kinetic
lability of radiometal complexes, the ultimate test for clinical
feasibility remains an in vivo study. This study demonstrates the
usefulness of HPG macromolecules to assess and compare the
physiologic stability of radiometal complexes over several days
in the bloodstream. Their high biocompatibility, chemical
robustness, and facile synthesis render HPG a suitable carrier
tool to investigate and compare long-term radiocomplex
stabilities in vivo.
CONCLUSIONS
■
The tetrakis(3,4-HOPO) chelator THPN and its newly
synthesized bifunctional derivative, p-SCN-Bn-THPN, were
investigated with zirconium(IV) for thermodynamic, in vitro,
and long-term in vivo stability. Solution studies revealed
exceptional thermodynamic stabilities with Zr and Fe that
are among the highest reported with these two metal ions.
Hyperbranched polyglycerol macromolecules were employed to
assess the long-term in vivo stability of Zr-THPN-HPG and
compare it against HPG-radioconjugates with the two literature
chelators DFO and DFO*. While all three radioconjugates
remained >95% intact in plasma in vitro, the in vivo study
revealed a lability of the THPN radioconjugate under
physiologic conditions evidenced by increasing bone uptake of
IV
III
8
9
Figure 6. PET images over 6 days of three mice injected i.v. with either
89
89
89
Zr-THPN-HPG (top), Zr-DFO-HPG (center), or Zr-DFO*-
8
9
Zr. The observed in vivo instability may be attributed to a small
HPG (bottom).
change in the coordination environment upon introduction of
the bifunctional linker, or to kinetic lability that may facilitate
demetalation under the highly dilute conditions encountered in
vivo.
While the precise mechanism for the observed instability
remains speculative, this study demonstrates the necessity for
such in vivo testing. It further informs the design of next-
generation chelators that may address potential limitations of
THPN such as by increasing kinetic inertness.
A precondition for the chosen testing methodology is the
knowledge and differentiability of the pharmacologic profile of
released radionuclide, such as the known bone-seeking proper-
8
9
4+
ties of Zr . If this premise is met, the use of large, stable
polymeric macromolecules is a suitable method to test and
compare the in vivo fate of radiochelates over several days. In
contrast to biomolecules such as mAbs that are often used for
such assessments, this method is not limited by requirements for
high specific activity, mild radiolabeling conditions, vector target
expression, or radiochelate density and solubility, which may
impair biomolecule integrity. Polymeric macromolecules may
therefore be a useful tool to investigate the in vivo fate of
radiochelates or other radiolabeled compounds.
Figure 7. 89Zr-bone uptake over time from the three radioconjugates
measured in tibias after 1, 3, or 6 days p.i. Results are shown as the mean
±
SD (n = 4).
thermodynamic stability but also on the kinetic inertness and the
metabolic stability, which can often have an even more profound
effect on the in vivo stability of metallo-radiopharmaceuti-
6
2−68
cals.
The importance of kinetic inertness is due to
diagnostic radiopharmaceuticals being administered in ex-
tremely low concentrations, which are further diluted as the
compounds enter circulation. Under these highly dilute
conditions, equilibrium conditions are no longer applicable
EXPERIMENTAL SECTION
■
Materials and Methods. Unless otherwise stated, all reagents and
solvents were purchased from commercial suppliers (Sigma-Aldrich,
TCI, AC PharmaChem, Acros, Strem Chemicals, Fisher Scientific) and
were used as received without further purification. All water used was
ultrapure (18.2 MΩ·cm) and was purified with a Millipore Milli-Q
and the rate of dissociation (k ) predominantly dictates in vivo
d
6
5−68
stability.
The radiometal complex, moreover, faces fierce
competition from orders of magnitude higher concentrations of
G
DOI: 10.1021/acs.inorgchem.9b02350
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
H
DOI: 10.1021/acs.inorgchem.9b02350
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Integral-10 purification system. Building block 6 (2-(aminomethyl)-
raphy strips (#150−005, black) developed with an aqueous DTPA
solution as mobile phase (50 mM, pH 7.0). Developed ITLC strips
were analyzed on a Packard Cyclone storage phosphor screen imager
with the OptiQuant software. Biodistribution samples were counted on
1
,6-dimethyl-3-(phenylmethoxy)-4(1H)-pyridinone) was purchased
69
from Otava Ltd. or was prepared according to a published procedure
with minor adjustments. p-SCN-Phe-DFO (p-SCN-Bn-deferoxamine)
1
2
was purchased from Macrocyclics, and p-SCN-Phe-DFO* (N -(8,19-
a calibrated PerkinElmer 2480 Wizard automated gamma counter.
scid
tm1Wjl
dihydroxy-1-((4-isothiocyanato-phenyl)amino)-9,12,20,23-tetraoxo-
NSG mice (NOD.Cg-Prkdc Il2rg
/SzJ) were obtained through an
1
4
1
(
-thioxo-2,8,13,19,24-pentaazanonacosan-29-yl)-N -hydroxy-N -(5-
N-hydroxyacetamido)pentyl)succinamide) was produced by Pharma
Inventor Inc. and was generously supplied by Dr. Benard’s lab (BC
Cancer Agency). Hyperbranched polyglycerol (HPG) was synthesized
in-house breeding program at the Animal Research Centre, BC Cancer
Research Centre. PET/CT images were acquired on a Siemens Medical
Solutions Inveon PET/CT small animal scanner.
́
Syntheses. Diethyl-2-(4-nitrobenzyl)malonate (1). This synthesis
5
3−55
47
and amino-functionalized according to published procedures.
brief, HPG of approximately 800 kDa (polydispersity index (PDI)
.23) was synthesized by ring-opening multibranching polymerization
In
was adapted from a literature procedure. Diethyl malonate (3.20 g,
20.0 mmol) was dropwise added to a suspension of sodium hydride
(60% in oil, 545 mg, 13.6 mmol) in anhydrous THF (7.5 mL) and was
stirred for 1 h at ambient temperature under argon. A solution of 4-
nitrobenzyl bromide (2.55 g, 11.8 mmol) in THF (7.5 mL) was
dropwise added while cooling in an ice bath. After complete addition,
the mixture was heated for 1.5 h at 35 °C. Once TLC indicated
completion of the reaction, the mixture was evaporated, taken up in
diethyl ether (20 mL), and washed with water (2 × 20 mL). The
0
of glycidol. Hydroxyl groups were oxidized with NaIO , followed by
4
reductive amination with 1,10-diaza-4,7-dioxadecane and NaCNBH3.
NMR spectra were recorded on a Bruker Ascend 400 spectrometer
1
13
(
(
(
400.13 MHz for H; 100.62 MHz for C) at 297.16 K. Chemical shifts
δ relative to residual solvent peak) are reported as parts per million
ppm) and coupling constants (J) in hertz (Hz). ESI-MS spectra were
recorded on an AB Sciex QTrap 5500 mass spectrometer. High-
resolution mass spectrometry (HR-MS) analyses were acquired by the
UBC Mass Spectrometry Centre on a Bruker HTCultra PTM
Discovery mass spectrometry system. Microwave reactions were
conducted in a Biotage Initiator+ microwave synthesizer. Hydro-
dynamic diameter measurements were carried out by dynamic light
scattering (DLS) on a Malvern Zetasizer Nano ZS. Measurements were
performed in triplicate in water at 25 °C using a 173° backscatter angle
and the material refractive index of polystyrene latex (1.590). Particle
size diameters are reported as Z-average (Z-avg. (d)). Data were
analyzed using the software Malvern Zetasizer (v. 7.12). HPLC was
performed on a Waters Alliance e2695 separations module coupled to a
Waters 2489 UV/vis-detector and, for radio-HPLC, a LabLogic Scan-
RAM radio-detector. For reversed phase HPLC, the column was a C18
Waters Atlantis T3, 100 Å, 5 μm particle size (4.6 × 150 mm),
supported by a C18 guard cartridge and was operated in an oven (40
organic phase was dried with MgSO , filtered, and evaporated.
4
Recrystallization from 1:1 Et O/hexane gave the title compound 1 as
2
1
yellow needles in 53% yield (1.84 g, 6.24 mmol). H NMR (400 MHz,
CDCl ): δ = 8.14 (d, J = 8.8 Hz, 2H, −Ph−H), 7.39 (d, J = 8.8 Hz, 2H,
3
−Ph−H), 4.23−4.10 (m, 4H, −O−CH −), 3.66 (t, J = 7.8 Hz, 1H,
2
−CH−), 3.31 (d, J = 7.6 Hz, 2H, −CH −Ph−), 1.21 (t, J = 7.2 Hz, 6H,
2
1
3
−CH ). C NMR (100 MHz, CDCl ): δ = 168.2, 146.9, 145.6, 129.8,
3
3
123.7, 61.8, 53.1, 34.3, 14.0. HPLC (method A): t = 15.9 min. ESI-MS:
R
+
−
m/z 318.2 [M + Na] ; 294.2 [M−H] .
2-(4-Nitrobenzyl)propanediamide (2). This synthesis was adapted
48
from a literature procedure. Compound 1 (1.64 g, 5.56 mmol) was
suspended in methanol (10 mL) and was placed in an ice bath. An
ammonia solution (30 mL, 7 N in methanol) was dropwise added to
this. The mixture was allowed to warm to ambient temperature and was
stirred overnight. HPLC indicated consumption of the starting material
and the presence of an intermediary product, which converted to the
product after stirring for another night at ambient temperature as
observed by HPLC. At this point, the suspension was filtered over
fritted glass and washed with methanol and boiling acetonitrile. Drying
°
C). Method A used the following gradient: A = 0.1% trifluoroacetic
acid (TFA) in water; B = methanol; flow rate = 1 mL/min; 0−5 min
0% A; 5−15 min 10−100% B; 15−18 min 100% B. Method B used the
9
following gradient: A = 0.1% trifluoroacetic acid (TFA) in water; B =
acetonitrile; flow rate = 1 mL/min; 0−5 min 90% A; 5−15 min 10−
1
in vacuo gave the title compound 2 as a white powder in 89% yield (1.18
1
g, 4.97 mmol). H NMR (400 MHz, DMSO-d ): δ = 8.14 (d, J = 8.8 Hz,
6
00% B; 15−18 min 100% B. Method C used the following gradient: A
2H, −Ph−H), 7.48 (d, J = 8.8 Hz, 2H, −Ph−H), 7.29 (s, 2H, −NH ),
2
=
10 mM (NH )HCO in water; B = 10 mM (NH )HCO in 2:1
7.09 (s, 2H, −NH ), 3.38 (t, J = 7.6 Hz, 1H, −CH−), 3.10 (d, J = 7.6
4
3
4
3
2
1
3
methanol/water; flow rate = 1 mL/min; 0−13 min 14−100% B; 13−18
min 100% B. Size-exclusion HPLC (SE-HPLC) was performed at
ambient temperature on the same radio-HPLC system but using a
Waters Ultrahydrogel Linear column (10 μm, 7.8 × 300 mm) eluted at
Hz, 2H, −CH −). C NMR (100 MHz, DMSO-d ): δ = 170.2, 148.0,
2
6
146.1, 130.2, 123.3, 54.0, 34.5. HPLC (method A): t = 11.46 min. ESI-
R
+
MS: m/z 260.0 [M + Na] .
1,3-Diamino-2-(4-nitrobenzyl)propane Dihydrochloride (3). This
48,70
0
.5 mL/min with an isocratic mobile phase consisting of a filtered
synthesis was adapted from reported procedures.
A dry round-
aqueous phosphate buffer (50 mM NaH PO , 50 mM Na HPO , 150
bottom flask was charged with compound 2 (129 mg, 542 μmol) and
was placed under nitrogen. To this was slowly added a solution of
2
4
2
4
mM NaCl, 10 mM NaN , pH = 6.2−7.0). Semipreparative HPLC was
3
performed on a Phenomenex Synergi Hydro-RP column (80 Å, 21.1 ×
borane stabilized in THF (BH ·THF, 1 M in THF, 1.2 mL, 1.2 mmol),
3
2
50 mm) connected to a Waters 600 controller and a Waters 2487 dual
and the mixture was stirred for 45 min at ambient temperature. Some
more anhydrous THF (5 mL) was added, and the mixture was heated at
reflux overnight. The reaction progress was monitored by HPLC, and
another aliquot of borane solution (0.2 mL) was added to the mixture.
It was heated for another night at reflux after which the reaction was
quenched by dropwise addition of concentrated HCl (2 mL). The
mixture was heated for an hour at reflux, and volatiles were evaporated
in vacuo. The yellow milky residue was taken up in water (3 mL), and at
0 °C, NaOH (6 M, 2.7 mL) was added using a dropping funnel. The
mixture was extracted with DCM (5 × 15 mL), and the organic phases
wavelength absorbance detector using the following gradient: A = 0.1%
TFA in water; B = methanol; flow rate = 10 mL/min; 5−100% B over
2
5 min. Flash chromatography was performed on a Biotage Isolera One
system using Biotage SNAP KP-Sil or ZIP silica gel cartridges (normal
phase) or Biotage SNAP Ultra C18 cartridges (reversed phase).
Analytical thin layer chromatography (TLC) was performed using silica
gel 60 F254 plates with aluminum backing obtained from Merck
Millipore. AW Standard Super-Cel filter aid was purchased from Sigma-
Aldrich. Semipreparative size-exclusion chromatography was per-
formed on PD MiniTrap G-25 desalting columns (Sephadex G-25,
GE Healthcare). Ultrafiltration was performed using Amicon Ultra
centrifugal filters of 100 kDa NMWL of 0.5 mL in volume (EMD
Millipore).
were combined, dried over MgSO , and evaporated. The resulting
4
yellow oil was suspended in ethanol (3 mL), and a drop of concentrated
HCl was added, the mixture was left to precipitate over 3 days at −20
°C. The supernatant was removed, and the precipitate was triturated
with diethyl ether and then dried in vacuo to give the title compound 3
8
9
Zr was obtained purified in 1 M oxalic acid from PerkinElmer
8
9
89
1
Health Sciences Canada, Inc. and was produced via the Y(p,n) Zr
as a light yellow powder in 66% yield (101 mg, 357 μmol). H NMR
reaction. 89Zr-oxalate solutions were neutralized with 2 M Na CO
(400 MHz, D O): δ = 8.23 (d, J = 8.8 Hz, 2H, −Ph−H), 7.50 (d, J = 8.8
2
3
2
solutions. Activities were measured using a Capintec CRC-55tR or a
Capintec CRC-25R/W dose calibrator. Instant thin-layer chromatog-
raphy (ITLC) was carried out using Biodex Tec-Control chromatog-
Hz, 2H, −Ph−H), 3.15 (dd, J = 13.6 Hz, 6.7 Hz, 2H, −N−CH −), 3.02
2
(dd, J = 13.6 Hz, 6.5 Hz, 2H, −N−CH −), 2.95 (d, J = 7.6 Hz, 2H,
2
1
3
−CH −), 2.57−2.46 (m, 1H, −CH−). C NMR (100 MHz, D O): δ
2
2
I
DOI: 10.1021/acs.inorgchem.9b02350
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
=
146.7, 145.2, 130.0, 124.0, 39.9, 36.6, 34.7. HPLC (method A): t =
173.4, 150.9, 149.9, 147.7, 147.3, 143.5, 138.4, 131.0, 130.0, 129.5,
R
+
6
.64 min. ESI-MS: m/z 210.0 [M + H] .
129.4, 124.6, 119.2, 74.6, 60.5, 60.4, 39.5, 37.9, 37.5, 36.2, 20.9. HPLC
+
Tetra-tert-butyl 2,2′,2′′,2′′′-((2-(4-nitrobenzyl)propane-1,3-diyl)-
(method B): t = 11.52 min. ESI-MS: m/z 1402.8 [M + H] , 1424.7 [M
R
+
−
bis(azanetriyl))-tetraacetate (4). Compound 3 (56 mg, 0.20 mmol)
was dissolved in anhydrous DMF (4 mL) and placed under N . To this
+ Na] ; 1400.8 [M − H] . HR ESI-MS: calcd (m/z) for
C H N O [M + H] 1402.6512; found 1402.6532 (1.4 ppm).
+ +
78 88 11 14
2
was added tert-butyl bromoacetate (129 μL, 0.87 mmol) and sodium
carbonate (∼0.39 g), and the mixture was stirred overnight at ambient
temperature. HPLC indicated completion of the reaction, and the
mixture was filtered and rinsed ad libitum with acetone. The filtrate was
evaporated and purified by flash chromatography over silica with 0−
2,2′,2′′,2′′′-((2-(4-Nitrobenzyl)propane-1,3-diyl)bis(azanetriyl))-
tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-
yl)methyl)acetamide) (8). Compound 7 (262 mg, 187 μmol) was
dissolved in glacial acetic acid (2.5 mL) and concentrated HCl (2.5
mL), and the mixture was stirred 24 h at 50 °C. The solution was
removed from heat, and a stream of nitrogen was blown over it
overnight before the residue was evaporated in vacuo. The residue was
taken up in a small amount of methanol (∼3.5 mL), precipitated by
dropwise addition to diethyl ether (∼40 mL), and placed in a freezer at
−20 °C for 20 min. The supernatant was removed and the precipitate
was dried in vacuo to give the title compound 8 as a white hydrochloride
2
4
0% ethyl acetate/hexane to give the title compound 4 as a yellow oil in
1
6% yield (60 mg, 90 μmol). H NMR (400 MHz, CDCl ): δ = 8.10 (d,
3
J = 8.8 Hz, 2H, −Ph−H), 7.41 (d, J = 7.6 Hz, 2H, −Ph−H), 3.35 (s, 8H,
−
−
−
N−CH −CO−), 2.88 (d, J = 6.0 Hz, 2H, −CH −Ph−), 2.74 (m, 2H,
2
2
N−CH −CH−), 2.47 (m, 2H, −N−CH −CH−), 1.96 (m, 1H,
2
2
1
3
CH−), 1.43 (s, 36H, −CH ). C NMR (100 MHz, CDCl ): δ =
3
3
1
1
70.6, 149.6, 146.1, 130.2, 123.3, 80.9, 56.6, 56.4, 38.3, 36.6, 28.1.
salt in ∼94% yield (243 mg, ∼176 μmol). H NMR (400 MHz,
HPLC (method A): t = 16.57 min. R = 0.37 (20% EtOAc/hexane).
CD
Hz, 2H, NO
N−CH
3
OD): δ = 8.19 (d, J = 8.0 Hz, 2H, NO −Ph−H), 7.65 (d, J = 7.6
2
R
f
+
+
ESI-MS: m/z 666.1 [M + H] , 688.1 [M + Na] .
2
−Ph−H), 7.18 (s, 4H, −CO−CH−), 4.81 (s, 8H, −CO−
),
2
,2′,2′′,2′′′-(2-(4-Nitrobenzyl)propane-1,3-diyl-bis(azanetriyl))-
2
−), 4.11 (sb, 8H, −N−CH
2
−CO−), 4.05 (s, 12H, −N−CH
3
tetraacetic Acid (5). Compound 4 (249 mg, 374 μmol) was dissolved
in 4.5 mL of acetonitrile, and ortho-phosphoric acid (∼1.5 mL, 85 wt %)
was dropwise added. This mixture was stirred one night at ambient
temperature and then heated another night at 45 °C. Once HPLC
indicated complete conversion, 3 mL of water was added, and the
mixture was concentrated in vacuo. The residual oil was taken up in
3.44−3.32 (sb, 4H, −N−CH −CH−), 3.04−2.94 (m, 1H, −CH−
2
CH −N−), 2.90−2.81 (m, 2H, −CH −Ph−NO ), 2.68 (s, 12H, −C−
2
2
2
1
3
CH ). C NMR (100 MHz, CD OD): δ = 168.6, 160.5, 150.7, 148.2,
3
3
147.2, 144.8, 140.3, 131.8, 124.8, 114.1, 61.1, 57.3, 40.5, 37.3, 36.1,
33.3, 21.4. HPLC (method A): t = 11.07 min. ESI-MS: m/z 1042.4 [M
R
+
+
+
+ H] , 1064.4 [M + Na] . HR ESI-MS: calcd (m/z) for C H N O
[M + H] 1042.4634; found 1042.4619 (1.4 ppm). Elemental analysis
50
64 11 14
+
(
NH )HCO (15 mL, 0.1 M), washed with ethyl acetate (10 mL), and
4
3
extracted with another 3 × 10 mL (NH )HCO3 solution. The
(%) calcd for C H N O ·7.4(HCl)·2.15(CH OH): C 45.36, H
4
50 63 11 14
3
combined aqueous layer was concentrated in vacuo and purified by
reversed phase chromatography (eluted with a gradient of 100% water
with 0.1% formic acid to 100% methanol). Fractions were pooled and
5.77, N 11.16; found C 45.55, H 5.54, N 10.94.
2,2′,2′′,2′′′-((2-(4-Aminobenzyl)propane-1,3-diyl)bis-
(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihy-
dropyridin-2-yl)methyl)acetamide) (9). In a Schlenk flask under inert
atmosphere, compound 8 (∼240 mg, ∼ 174 μmol) was dissolved in
methanol (∼20 mL), and Pd/C was added (10% w/w, 19.6 mg, 18.4
μmol). The reaction vessel was sealed and placed under a hydrogen
atmosphere (balloon), and the mixture was stirred vigorously at
ambient temperature. After 2.5 h, the reaction mixture was vented to
nitrogen, filtered through a plug of filter aid, and generously rinsed with
methanol. The filtrate was purified by reversed phase flash
chromatography (eluting with 0−100% methanol/water with 0.1%
formic acid). Product fractions were pooled, evaporated, and dried in
vacuo to give the title compound 9 in its formic acid salt as an off-white
evaporated to give the title compound 5 as a fluffy white powder in 97%
1
yield (173 mg, 364 μmol). H NMR (400 MHz, CD OD): δ = 8.17 (d, J
3
=
8.8 Hz, 2H, −Ph−H), 7.51 (d, J = 8.8 Hz, 2H, −Ph−H), 3.81 (d, J =
1
6.8 Hz, 4H, −N−CH −CO−), 3.68 (d, J = 16.8 Hz, 4H, −N−CH −
2
2
CO−), 3.48 (dd, J = 13.2, 3.6 Hz, 2H, −N−CH −CH−), 3.09−3.03
2
(
2
1
m, 2H, −N−CH −CH−), 2.75 (d, J = 7.2 Hz, 2H, −CH −Ph−), 2.7−
2
2
13
.6 (m, 1H, −CH−). C NMR (100 MHz, CD OD): δ = 172.8, 148.2,
48.0, 131.3, 124.7, 59.6, 56.8, 37.6, 33.4. HPLC: t = 11.81 min. ESI-
3
R
+
+
MS: m/z 442.1 [M + H] , 464.2 [M + Na] . Elemental analysis (%)
calcd for C H N O ·H O·1/2(CH OH): C 46.77, H 5.72, N 8.84;
18
23
3
10
2
3
found C 46.97, H 5.77, N 9.01.
1
2
,2′,2′′,2′′′-((2-(4-Nitrobenzyl)propane-1,3-diyl)bis(azanetriyl))-
solid in ∼99% yield (∼188 mg, ∼173 μmol). H NMR (400 MHz,
tetrakis(N-((1,6-dimethyl-3-(phenylmethoxy)-4-oxo-1,4-dihydro-
pyridin-2-yl)methyl)acetamide) (7). A microwave vial was charged
with compound 5 (108 mg, 226 μmol), which was suspended in
CD OD): δ = 6.75 (d, J = 8.4 Hz, 2H, −Ph−H), 6.64 (d, J = 8.0 Hz, 2H,
3
−Ph−H), 6.35 (s, 4H, −CO−CH−), 4.61 (s, 8H, −CO−N−CH −),
2
3.66 (s, 12H, −N−CH ), 3.19 (s, 8H, −N−CH −CO−), 2.53−2.48
3
2
anhydrous DMF (4 mL). To this was added HOBt·H O (173 mg, 1.13
(m, 2H, −N−CH −CH−), 2.36 (s, 12H, −C−CH ), 2.40−2.31 (m,
2
2
3
mmol, 5.0 equiv) and a solution of DCC (233 mg, 1.13 mmol, 5.0
equiv) in DMF (4.5 mL). The mixture was stirred for 2 h at ambient
2
H, −N−CH −CH−), 2.40−2.31 (m, 2H, −CH −Ph−), 1.64−1.59
2
2
13
(m, 1H, −CH−CH −N−). C NMR (100 MHz, CD OD): δ = 173.7,
2
3
temperature under N . At this point, 2-(aminomethyl)-1,6-dimethyl-3-
2
170.6, 148.8, 147.3, 144.2, 132.7, 132.4, 130.7, 117.9, 114.8, 61.0, 60.5,
(
phenylmethoxy)-4(1H)-pyridinone (6, 292 mg, 1.13 mmol, 5.0 equiv)
3
9.6, 37.4, 37.4, 35.8, 21.0. HPLC (method A): t = 9.79 min. ESI-MS:
R
+
+
was added; the mixture was rinsed with another 2 mL of DMF, and the
m/z 1012.5 [M + H] , 1034.5 [M + Na] . HR ESI-MS: calcd (m/z) for
C H N O [M + H] 1012.4892; found 1012.4908 (1.6 ppm).
+
+
vial was crimp capped under N . The mixture was heated in a
2
5
0
66 11 12
microwave reactor for 4 h at 55 °C and then stirred at ambient
temperature overnight. The mixture was heated another 2 h at 55 °C in
the microwave reactor and then stirred a second night at ambient
temperature. At this point, HPLC indicated complete conversion, and
the mixture was filtered over a cotton plug, rinsed with acetonitrile,
evaporated, and purified by reversed phase flash chromatography
2
,2′,2′′,2′′′-((2-(4-Isothiocyanatobenzyl)propane-1,3-diyl)bis-
(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihy-
dropyridin-2-yl)methyl)acetamide) (p-SCN-Bn-THPN, 10). A solution
of 9 (∼116 mg, ∼115 μmol) in water (∼10 mL) was added to a solution
of thiophosgene (131.8 μL, 1.719 mmol, 15 equiv) in CHCl (1.5 mL).
3
Caution! Thiophosgene is highly toxic and special care must be exercised
in its handling.
(
eluted with a gradient of 100% water with 0.1% formic acid to 40%
acetonitrile). Product fractions were pooled and concentrated in vacuo
The reaction mixture was stirred vigorously overnight at ambient
temperature. The mixture was transferred with water (5 mL) to a
to give the title compound 7 as a yellow oil in ∼83% yield (262 mg, 187
1
μmol). H NMR (400 MHz, CD OD): δ = 8.01 (d, J = 8.4 Hz, 2H,
conical centrifuge tube, washed with CHCl (4 × 1 mL) by biphasic
3
3
NO −Ph−H), 7.41−7.39 (m, 8H, Bn−H), 7.29−7.26 (m, 12H, Bn−
stirring using a vortex, and had the organic layers removed with a pipet.
2
H), 7.16 (d, J = 8.8 Hz, 2H, NO −Ph−H), 6.40 (s, 4H, −CO−CH−),
The aqueous layer was diluted with H O to ∼25 mL and was purified in
2
2
5
1
2
1
.14 (s, 8H, −O−CH −Ph), 4.42 (s, 8H, −CO−N−CH −), 3.53 (s,
separate batches either by semipreparative HPLC as described above or
by reversed phase flash chromatography (eluted with a gradient of 5−
70% methanol with 0.1% TFA/water with 0.1% TFA). Product
fractions were pooled, concentrated by rotary evaporation, and dried
overnight by lyophilization to yield the title compound 10 (p-SCN-Bn-
2
2
2H, −N−CH ), 3.02 (s, 8H, −N−CH −CO−), 2.54 (d, J = 6.4 Hz,
3
2
H, −CH −Ph−NO ), 2.47−2.43 (m, 2H, −N−CH −CH−), 2.33 (s,
2
2
2
2H, −C−CH ), 2.21−2.16 (m, 2H, −N−CH −CH−), 1.64−1.57
3
2
1
3
(
m, 1H, −CH−CH −N−). C NMR (100 MHz, CD OD): δ = 174.7,
2
3
J
DOI: 10.1021/acs.inorgchem.9b02350
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
1
THPN) as a white powder in ∼65% yield (∼79 mg, ∼75 μmol). H
NMR (400 MHz, CD CN/D O, 11:1 v/v): δ = 7.22 (d, J = 8.4 Hz, 2H,
week-old female, healthy, immunodeficient NSG mice (NOD.Cg-
scid
tm1Wjl
Prkdc Il2rg
/SzJ) were anesthetized using isoflurane inhalation
3
2
8
9
−
Ph−H), 7.16 (d, J = 8.4 Hz, 2H, −Ph−H), 7.00 (s, 4H, −CO−CH−),
(2% in O at 2 mL/min) and were administered Zr-THPN-HPG,
2
8
9
89
4
3
.61−4.52 (m, 8H, −CO−N−CH −), 3.79 (s, 12H, −N−CH ), 3.69−
Zr-DFO-HPG, or Zr-DFO*-HPG conjugate solutions via tail vein
injection.
2
3
.59 (m, 8H, −N−CH −CO−), 3.01−2.97 (m, 2H, −N−CH −
2
2
CH−), 2.85−2.79 (m, 2H, −N−CH −CH−), 2.48 (s, 12H, −C−
For biodistribution studies, each mouse was injected ∼1.5 MBq
2
89
CH ), 2.46−2.43 (m, 2H, −CH −Ph−), 2.32−2.27 (m, 1H, −CH−
(∼40 μCi, median, ∼ 40 μL) Zr-chelate-HPG solution and mice were
3
2
13
CH −Ph−). C NMR (100 MHz, CD CN/D O, 11:1 v/v): δ = 170.3,
euthanized after 1, 3, or 6 days p.i. by CO inhalation, followed by blood
2
3
2
2
1
5
61.9, 150.4, 144.4, 139.5, 138.9, 131.3, 130.0, 126.8, 116.1, 114.3, 60.8,
7.1, 39.6, 36.6, 36.0, 33.8, 21.4. HPLC (method C): t = 13.15 min.
withdrawal via cardiac puncture (n = 4 per group and time point).
Tissues and organs were harvested, washed in Dulbecco’s PBS, dried on
R
2
+
+
+
89
ESI-MS: m/z 527.8 [M + 2H] , 1054.4 [M + H] , 1076.4 [M + Na] .
paper, and weighed. Zr activities were measured using a calibrated γ
+
+
HR ESI-MS: calcd (m/z) for C H N O S [M + H] 1054.4457;
counter. Data were decay corrected, and activity distributions were
expressed as percentage of the injected dose per gram of tissue (%ID/
g). These results were compiled in Microsoft Excel and evaluated in
GraphPad Prism and Excel. Outliers were identified via a Grubbs test (p
5
1
64 11 12
found 1054.4493 (3.6 ppm).
Chelator Conjugation to HPG. THPN-, DFO-, and DFO*-HPG
Conjugates. In three separate reaction vials, aqueous solutions (100
μL, 40 g/L, ∼5 nmol) of amine-functionalized hyperbranched
polyglycerol (HPG, ∼800 kDa molecular weight) were diluted with
sodium carbonate buffer (150 μL, 0.1 M, pH 9), and ligand solutions of
either p-SCN-Bn-THPN, p-SCN-Phe-DFO, or p-SCN-Phe-DFO*
were added (50 μL, 0.5 M, ∼ 5 equiv). DFO and DFO* stock
solutions were in anhydrous DMSO, while the THPN stock solution
was in freshly diluted 5% DMSO (aq). The pH of the reaction mixtures
71
<
0.01) using the software R and were excluded from the reported
results. Statistical analysis of bone uptake was performed by multiple t-
tests assuming equal variance with a significance level of α = 0.05.
Activity distributions are expressed as percentage of the injected dose
per gram of tissue (%ID/g).
For PET/CT imaging, each mouse was injected ∼5 MBq (∼135 μCi,
8
9
∼
120 μL) of Zr-chelate-HPG solution (n = 1 per group). Prior to
was adjusted to pH 8.5−9.0 with Na CO , and the mixtures were
2
3
imaging, mice received a subcutaneous injection of 0.9% NaCl (0.2 mL)
incubated overnight at 37 °C with gentle agitation (350 rpm). After 21
h, the mixtures were loaded on a PD MiniTrap G-25 size-exclusion
column, eluted with HEPES buffer (0.5 M, pH 7.0), and further purified
by ultrafiltration over Amicon Ultra 100 K centrifugal filters (rinsed
with 3 × 0.4 mL HEPES buffer). The recovered HPG conjugates were
analyzed by SE-HPLC and DLS.
for hydration during imaging. Mice were imaged under isoflurane
anesthesia (2% in O at 2 mL/min) with a CT scan followed by a 20 min
2
PET acquisition at 1 h, 1 d, 3 d, and 6 d p.i. After the last imaging time
point, mice were euthanized by CO inhalation followed by blood
2
withdrawal by cardiac puncture and were included in the respective
biodistribution studies described above. Images were reconstructed
using a three-dimensional ordered subsets expectation maximization
(OSEM3D, 2 iterations), followed by a fast maximum a priori algorithm
(FastMAP, 18 iterations), and attenuation correction was performed
based on the CT image. Images were rendered using the Inveon
Research Workplace software.
THPN-HPG, DFO-HPG, DFO*-HPG, HPG-NH (Control). SE-HPLC:
2
tR = 15.3 min, 15.4 min, 15.4 min, 15.2 min. DLS: Z-avg. (d) 15.4 ± 0.5
nm, 19.2 ± 0.6 nm, 16.9 ± 0.4 nm, 16.7 ± 0.2 nm. PDI: 0.15 ± 0.04,
0
.32 ± 0.05, 0.23 ± 0.02, 0.23 ± 0.01.
89
Zr-Labeling of HPG-Chelator Conjugates. In three separate
reaction vials, HPG-chelator solution (THPN, DFO, or DFO*) was
Thermodynamic Solution Studies. General. Protonation
8
9
added to a neutralized Zr-oxalate solution (∼170 μL, ∼32 MBq, 870
μCi, pH ∼ 7), it was briefly vortexed, and the mixture was incubated at
constants and metal stability constants were obtained by combined
72
potentiometric−spectrophotometric titrations as described before.
2
1 °C with gentle agitation (350 rpm). Aliquots were analyzed by
Measurements were conducted at 25 °C and an ionic strength of I =
0.16 M NaCl, using a Metrohm Titrando 809 equipped with a Ross
combined electrode, a Metrohm Dosino 800, and a Varian Cary 60
UV−vis spectrophotometer (200−800 nm spectral range) connected
to a 0.2 cm path length (l) optic dip probe immersed in the titration cell.
Additional batch experiments to study proton competition or metal−
metal competition were carried out in a 1 cm cuvette at 25 °C and 0.16
M NaCl ionic strength to determine the formation constants of the
ITLC, and more HPG-chelator solution was added in portions until
quantitative radiolabeling was achieved. Added HPG-chelate volumes
were 67.4, 75.6, and 66.6 μL for the THPN, DFO, and DFO*
conjugates, respectively, and the total reaction volume was ∼250 μL for
each reaction. After a total of 3 h reaction time, the mixtures were
purified by size exclusion chromatography (PD MiniTrap G-25) and
eluted with water in fractions. Isolated radiochemical yields for all three
reactions were 97%, and radiochemical purities were >99% (HPLC).
Specific activities were estimated as ∼9.6, ∼10.2, and ∼11.5 MBq/mg
for the THPN, DFO, and DFO* radioconjugates, respectively.
III
IV
Fe -THPN and Zr -THPN systems. Metal ion solutions were
prepared from atomic absorption (AA) standard metal ion solutions.
The exact amount of acid present in the iron and zirconium standards
8
9
89
89
73
Zr-THPN-HPG, Zr-DFO-HPG, Zr-DFO*-HPG. Radio-SE-
HPLC: t = 15.3 min, 15.3 min, 15.3 min. DLS: Z-avg. (d) 16.2 ±
was determined by Gran’s method, titrating equimolar solutions of
R
either metal ion and Na H EDTA. All potentiometric measurements
2
2
7
4
1
.0 nm, 14.8 ± 0.2 nm, 18.3 ± 0.7 nm. PDI: 0.25 ± 0.02, 0.22 ± 0.01,
were processed using the Hyperquad2013 software, whereas
spectrophotometric data were processed using the HypSpec2014
software. Speciation plots for the ligand and metal complexes were
0.2I 9n ±V i0 t. r0 o3 .Plasma Stability Study. 8
9Zr-chelate-HPG conjugates
33
75
with THPN, DFO, or DFO* prepared as described above were diluted
calculated with HySS using the constants in Tables 1 and 2 and the
metal hydrolysis equilibrium constants. The molar absorptivities of all
protonated species of THPN were included in the metal stability
calculations. Proton dissociation constants corresponding to hydrolysis
1
:4 (v/v) with human blood plasma from a healthy donor. The mixtures
were incubated for 5 days at 37 °C with gentle agitation (350 rpm).
Aliquots were analyzed by ITLC at the beginning of the experiment and
8
9
3+
4+
after 1, 3, and 5 days incubation, and the percentage of chelated Zr was
determined by integration. Measurements were performed in
triplicates.
of aqueous Fe and Zr 7 ions included in the calculations were taken
6
from Baes and Mesmer. The species formed in the studied systems
were characterized by the general equilibrium: mM + lL + hH ⇆
In Vivo Study of 89Zr-HPG Conjugates. This animal study was
performed in accordance with the animal care committee (ACC) of the
University of British Columbia under the approved protocol A16-0104.
M L H (charges omitted). For convention, a complex containing
m
l
h
metal ion M, ligand L, and proton H has the general formula M L H .
m
l
h
The stoichiometric index m might also be 0 in the case of protonation
equilibria. Negative values for the index h refer to proton removal or
hydroxide ion addition during formation of a M−OH complex. The
overall equilibrium constant for the formation of the complex M L H
h
89
Zr-chelate-HPG conjugates with THPN, DFO, or DFO* chelators
were prepared as described above on the day prior to the experiment
and were stored overnight at 4 °C. On the day of administration, quality
control of the radioconjugates was performed by radio-SE-HPLC and
ITLC. Immediately before preparation of the injection doses, the
aqueous radioconjugate stock solutions (∼490 μL) were diluted with
sterile-filtered (0.22 μm) concentrated PBS (10x, 54.4 μL). Eleven-
m
l
from its components is designated as log β. Stepwise equilibrium
constants log K correspond to the difference in log units between the
overall constants of sequentially protonated (or hydroxide) species. pM
n+
n+
is defined as −log [M ] and is always calculated at [M ] = 1 μM,
free
K
DOI: 10.1021/acs.inorgchem.9b02350
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
[
Lx−] = 10 μM, pH 7.4, and 25 °C.41 To facilitate reading, ligand
AUTHOR INFORMATION
■
nomenclature is generally described using the neutral form THPN
herein without reflecting its protonation state unless specific
protonation of the ligand is being discussed.
THPN Ligand Protonation Constants. Protonation equilibria of the
ligand were studied by simultaneous potentiometric−spectrophoto-
metric titrations of an acidic solution of THPN (8.47 × 10− M) at 25
Corresponding Authors
*E-mail: urs.hafeli@ubc.ca (U.O.H.).
*E-mail: kathy.saatchi@ubc.ca (K.S.).
*E-mail: buchwalder@alumni.ubc.ca (C.B.).
5
ORCID
°
C, l = 0.2 cm, and 0.16 M NaCl ionic strength. Electromotive force
values and spectra were recorded after each addition of NaOH (0.1496
M) or HCl, and both instruments were synchronized in order to have
constant delays between each titrant addition and to allow enough time
to reach equilibrium. The potentiometric and spectrophotometric data
collected for at least three replicates were processed using the
Christian Buchwalder: 0000-0001-5577-8870
María de Guadalupe Jaraquemada-Pelaez: 0000-0002-6204-
́
707X
Cristina Rodríguez-Rodríguez: 0000-0002-3313-4422
Chris Orvig: 0000-0002-2830-5493
33,74
HypSpec2014 and Hyperquad2013 software suites.
molar absorptivities of the differently protonated species were also
determined.
Metal Complex Formation Constants. The strength of the resulting
metal complexes made it necessary to employ different methods to
study the complexation of THPN with Fe and Zr . UV−vis
experiments were conducted to determine the Fe -THPN formation
constants by direct proton competition and to study the Fe /Zr
competition. In addition to these experiments, direct simultaneous
The respective
Franco
Urs O. Haf
̧
is Ben
́
ard: 0000-0001-7995-3581
̈
eli: 0000-0003-0671-4509
Author Contributions
All authors have given approval to the final version of the
manuscript.
III
IV
III
III
IV
Notes
The authors declare no competing financial interest.
potentiometric−spectrophotometric titrations were carried out with
III
IV
both the Fe -THPN and the Zr -THPN systems once their first
formation constants were determined using the above methods.
Direct proton competition experiments were carried out by UV-
potentiometric batch experiments (spectral range = 200−800 nm, 25
ACKNOWLEDGMENTS
■
Funding for this research is gratefully acknowledged from the
Natural Sciences and Engineering Research Council of Canada
NSERC, Discovery Grant no. 312284 to U.O.H., Discovery
Grant no. RGPIN-2018-04772 to C.O., and CREATE IsoSiM
fellowship to C.B.), the University of British Columbia (Four
Year Doctoral Fellowship to C.B.), and the NSERC and CIHR
3
+
°
7
C, l = 1 cm) on a set of 22 solutions containing [THPN] = [Fe ] =
(
.16 × 10− M with different amounts of standardized HCl to cover a
5
pH range of 0.16−3.26. The ionic strength of each sample was adjusted
to 0.16 M by addition of different amounts of NaCl.
2
+
To determine the formation constant of the [Zr(H THPN)]
2
(
Canadian Institutes of Health Research) for Collaborative
complex, metal−metal competition UV−vis titrations were carried
3
+
−5
out. A set of 20 solutions containing [THPN] = [Fe ] = 7.16 × 10
M
Health Research Project support to F.B., C.O., and P.S. (CPG-
146482 (CIHR) and CHRP 493725-16 (NSERC)). This study
was further supported by the Lundbeck Foundation, Denmark
(grant no. 2014-4176, the UBC-SUND Lundbeck Foundation
professorship to U.O.H.). Computational resources for the DFT
calculations are acknowledged from WestGrid and Compute
Canada.
4
+
with varying amounts of competing Zr ions were prepared with
4+
3+
[
Zr ]/[Fe ] molar ratios ranging from 0 to 4.5. The pH was adjusted
with standardized HCl to pH 2 and the ionic strength of each sample
was adjusted to 0.16 M by addition of different amounts of NaCl as
supporting electrolyte. The samples were left up to 24 h in order to
reach equilibrium before measurement of pH and UV−vis spectra.
UV−vis measurements were carried out in a 1 cm cuvette at 25 °C and
2
50−700 nm spectral range.
Additional direct UV-potentiometric titrations were carried out for
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III
IV
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