H2hox: Dual-Channel Oxine-Derived Acyclic Chelating Ligand for 68Ga Radiopharmaceuticals

Article abstract of DOI:10.1021/acs.inorgchem.8b01208

An acyclic hexadentate oxine-derived chelating ligand, H₂hox, was investigated as an alternative to current chelators for ⁶⁸Ga. The straightforward preparation of H₂hox, involving only one or two steps, obviates the synthe

Full text of DOI:10.1021/acs.inorgchem.8b01208

Article
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/IC
H hox: Dual-Channel Oxine-Derived Acyclic Chelating Ligand for
2
6
8
Ga Radiopharmaceuticals
†
z,† Yang Cao,† Jinhe Pan, Kuo-Shyan Lin,‡
‡
Xiaozhu Wang, María de Guadalupe Jaraquemada-Pelae
́
†
,†
Brian O. Patrick, and Chris Orvig*
†Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver,
British Columbia V6T 1Z1, Canada
‡
BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada
*
S Supporting Information
ABSTRACT: An acyclic hexadentate oxine-derived chelating ligand, H hox, was
2
6
8
investigated as an alternative to current chelators for Ga. The straightforward
preparation of H hox, involving only one or two steps, obviates the synthetic
2
68
3+
challenges associated with many reported Ga chelators; it forms a Ga complex
of great stability (log K = 34.4) with a remarkably high gallium scavenging ability
3
+
3+
3+
x−
(
°
pGa = −log[Ga
] = 28.3, ([Ga ] = 1 μM; [L ] = 10 μM; pH 7.4, and 25
free
68
C)). Moreover, H hox coordinates Ga quantitatively in 5 min at room
temperature in ligand concentrations as low as 1 × 10 M, achieving an
unprecedented high molar activity of 11 ± 1 mCi/nmol (407 ± 3.7 MBq/nmol)
without purification, suggesting prospective kit-based convenience. [ Ga(hox)]
showed no decomposition in a plasma challenge. Good in vivo stability and fast
renal and hepatic clearance of the [ Ga(hox)] complex were demonstrated using
dynamic positron emission tomography/computed tomography imaging. The
2
−
7
6
8
+
68
+
+
intrinsic fluorescence of [Ga(hox)] allowed for direct fluorescence imaging of
cellular uptake and distribution, demonstrating the dual-channel detectability and intracellular stability of the metal complex.
INTRODUCTION
postsynthetic purification steps and workup for the removal of
■
13
6
8
68
impurities.
The Ge/ Ga generator system is one of the most attractive
systems for diagnostic nuclear medicine. It could be utilized for
an extended period due to the 271 d half-life of the parent
Over the past decade, several Ga3+ chelators have been
developed to overcome these drawbacks including 1,4,7-
1
4,15
16
6
8
68
triazacyclononane-1,4,7-triacetic acid (NOTA),
NOTP,
and (1,2-[[6-
isotope Ge. The daughter isotope Ga has a high positron
decay yield (89%, 1.899 keV) and a relatively short half-life
time (t1/2 = 68 min), and it is becoming increasingly important
as a versatile and easily available positron emission tomography
17,18
19−21
22
23−25
TRAP,
DATA,
PCTA, HBED,
1
,26−32
carboxypyridin-2-yl]methylamino]-ethane (H
dedpa)
2
3
+
(Scheme 1). NOTA radiolabels Ga at RT in 10 min at pH
3−5.5. NOTP and TRAP, the triphosphate analogues of
NOTA, could be used in a broader pH range, especially at
HBED and its bioconjugate showed a high
thermodynamic stability and serum stability. THP achieved a
7% radiochemical yield at 0.5 μM and near neutral pH, and
1
−3
14
(
PET) imaging tracer.
Because of the 68Ga short half-life, an ideal chelating ligand
16,33
should rapidly achieve efficient and reproducible radiolabeling
under mild conditions (room temperature (RT) and near
neutral pH), yielding a stable radiopharmaceutical with high
molar activity at low concentration that could be used in
routine clinical practice without further purification. Thus,
such a radiopharmaceutical ligand would be ideal for the
lower pH.
23
7
9
finally H dedpa can obtain a quantitative radiochemical yield at
2
27
concentrations as low as 0.1 μM.
There are still limitations among those chelators. For
example, the labeling performance of NOTA and TRAP at
near-physiological pH is not as good as at lower pH and
requires higher concentrations; HBED forms multiple species
4
−7
development of a toolkit for convenient clinical use.
The
macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid (DOTA; Scheme 1) is the most commonly
6
8
8−12
used chelator for Ga labeling.
The preorganized closed
3
+
in solution with Ga , and thus, it is not ideal for quick and
chain structure guarantees a high kinetic inertness and
thermodynamic stability; however, concomitant are longer
labeling time and high temperature, which is incompatible with
the labeling of thermally sensitive biovectors. Moreover, the
synthesis of those cyclic chelators and corresponding bifunc-
tional derivatives is also challenging and expensive involving
7
easy purification and kit-based application. The synthesis of
19
THP and its derivative are nontrivial. Synthetic accessibility
Received: May 3, 2018
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.8b01208
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Scheme 1. Examples of Chelating Ligands for Ga3+ from the
Literature
successful, showing fast chelation and high stability, properties
close to those of DOTA and NOTA, until we discovered
27,34−38
H hox.
The high stability of H dedpa was attributed to
2
2
a nearly perfect size fit and geometric arrangement of
coordination atom and bonds. Oxine (8-hydroquinoline), the
bidentate ligand in the anticancer compound KP46, possesses a
very high stability constant for its 3:1 complex with gallium
39
(
log βML3 = 36.4). In the design of our second generation of
acyclic chelators for gallium, a combination of the respective
advantages of H dedpa and oxine was sought, leading to the
2
structure of H hox (Scheme 2).
2
Synthesis and Characterization. The only previous
40,41
report of H hox was in 1972 by Hata and Uno
as an
2
analytical reagent (BHQED) for divalent metals, and it has not
been further explored in the intervening near-half-century;
however, Hata and Uno’s synthetic protocol was difficult to
reproduce, resulting in low yield and purity. Thus, a modified
synthetic route was developed (Scheme 3). 8-Hydroxyquino-
a
Scheme 3. Synthesis of H hox
2
a
(
a) Ethylenediamine, CH CH OH, at 60°C, 4 h; CH CH OH,
3 2 3 2
NaBH (5 equiv), overnight.
4
is also a challenge for H dedpa and most of the cyclic chelators
2
2
7
as well.
In this work, we report a tightly binding acyclic hexadentate
chelator that will certainly be applicable to Ga PET imaging:
line-2-aldehyde is a cheap starting material purchased from a
commercial supplier or synthesized from 2-methyl-8-hydrox-
68
yquinoline in one step without protection. H hox was
2
H hox with rigid “bis-ox” (8-hydroxyquinoline) arms (Scheme
2
synthesized by reductive amination of 8-hydroxyquinoline-2-
aldehyde and ethylenediamine (en) in one step and purified by
recrystallization to obtain 87% yield without the need of
column separation. In the Hata and Uno synthetic protocol,
most of the product precursor was filtered out and discarded
before the reaction was quenched with acetic acid, giving a low
yield. Therefore, in our new protocol the reaction was
quenched by HCl directly, and the product precipitated out
in high yield after the pH was adjusted to neutral. The new
route takes only one or two steps (depending on starting
material), quickly and economically yielding grams of ligand.
This is particularly notable when comparing with most of the
previously reported ligands such as NOTA, DOTA, TRAP,
2
). The synthesis of H hox is easy and fast (one or two steps),
2
Scheme 2. Design Paradigm for H hox
2
and solution studies reveal the presence of a single complex
species in a broad pH range (1−11) with higher log K
NOTP, DATA, THP, and H dedpa. Moreover, in work to be
ML
2
3
+
(
34.4) and pM value (28.3) than for most of the Ga
published subsequently, we can conveniently form a bifunc-
tional version by using a functionalized diamine backbone
(e.g., (4-nitrobenzyl)ethylenediamine). The bifunctional tracer
and the hydrophilicity tuning can be achieved by direct
modification on the aromatic ring; this has been widely used in
the 8-hydroquinoline-based pharmaceuticals and in organic
68
chelators. H hox showed fast and quantitative Ga complex-
2
ation at mild conditions (5 min, RT) with a concentration as
−
7
low as 1 × 10 M and an unprecedented high molar activity
without purification. Plasma challenge experiments and
dynamic PET imaging confirm excellent in vitro and in vivo
stability. These characteristics suggest strong relevance to
toolkit radiopharmaceuticals (“shake and shoot”); to comple-
ment these ideal properties, the florescence of the chelating
ligand is turned on by complexation to Ga , and this was used
to investigate the cellular distribution of [Ga(hox)] directly
and showed the potential for dual-channel or bimodal imaging.
42−46
light emitting diode (OLED) agents.
The tedious and
challenging synthesis and purification of most of the previous
chelators is a significant barrier to commercial applicability.
3+
H hox overcomes this barrier and is ideally suited for real
2
+
clinical research and wide application.
X-ray Crystallography and DFT Calculations. The
structure of H hox was confirmed by X-ray diffraction data
using a suitable crystal obtained by recrystallization from
methanol. Two crystallographically independent structures,
2
RESULTS AND DISCUSSION
Our group has screened many acyclic chelators for gallium
radiolabeling in the past decade, and H dedpa was the most
■
both with C symmetry (Figure S7), were identified featuring
2
i
B
DOI: 10.1021/acs.inorgchem.8b01208
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Inorganic Chemistry
Article
two 8-hydroxyquinoline moieties connected to an ethylenedi-
amine backbone giving rise to six potential coordination sites
(
Figure 1a). [Ga(hox)][ClO ] crystals were obtained by layer
4
Figure 1. ORTEP diagrams of (a) one of the two crystallographically
independent structures of H hox, and (b) two crystallographically
2
+
Figure 2. DFT-optimized structures of the (a) [Ga(hox)] and (b)
independent cations of [Ga(hox)] [ClO ]; thermal ellipsoids shown
4
+
[
Ga(dedpa)] cations with hexacoordinated metal centers, and the
at 50% probability level; hydrogen atoms and solvent acetonitrile
molecules omitted for clarity.
electrostatic potentials of (c) [Ga(hox)] and (d) [Ga(dedpa)]+
mapped onto their electron density. The MEP represents a maximum
potential of 0.25 au and a minimum of −0.02 au mapped onto
+
diffusion of diethyl ether into a dichloromethane solution of
the complex, and the material crystallizes with two Ci
symmetric structures (Figure 1b). Selected bond parameters
−
3
electron density isosurface (0.002 e Å , red to blue = negative to
positive).
+
of the mer-[Ga(hox)] cation are summarized in Table 1 and
Solution Thermodynamics. High thermodynamic stabil-
ity (log KML) and kinetic inertness are required for in vivo
application of radiopharmaceutical metal complexes, to
minimize transchelation and transmetalation reactions caused
by endogenous ligands and metals. The stepwise protonation
Table 1. Selected Bond Distances in the Cations [Ga(hox)]+
+
and [Ga(dedpa)]
a
[
Ga(hox)]+
[Ga(dedpa)]+
atom
constants of H hox included in the calculation of the log K
atom
atom
length (Å)
atom
length (Å)
2
ML
were determined by UV in batch spectrophotometric titrations,
as the pH-potentiometric method was unsuitable because of
Ga1
Ga1
Ga1
Ga1
Ga1
Ga1
O11
O1
N1
N11
N21
N2
1.959
1.959
1.982
1.982
2.190
2.190
Ga1
Ga1
Ga1
Ga1
Ga1
Ga1
O11
O1
N1
N11
N21
N2
1.970
1.982
1.986
1.989
2.112
2.113
the insufficient solubility of H hox. The spectral data were
2
47
refined using the HypSpec2014 program (Table 2).
Table 2. Protonation Constants of H hox at 25°C
2
a
a
From ref 27; pyr = pyridine, and en = ethylenediamine.
equilibium
log β
log K
log K
+
b
b
b
b
c
L + H ⇆ HL
10.88(1)
20.69(1)
29.08(1)
35.14(2)
35.78(6)
36.02(8)
10.88
11.35
10.81
8.15
5.12
ND
+
+
b
HL + H ⇆ H L
H L + H ⇆ H L
H L + H ⇆ H L
H L + H ⇆ H L
9.81
compared to those of mer-[Ga(dedpa)] . The meridional
2
+
+
b
8.39
arrangement of the six-coordinating atoms in [Ga(hox)] is
2
3
+
+
+
b
6.06
0.64
similar to that in [Ga(dedpa)] ; however, in [Ga(hox)] , the
Ga−O and Ga−N(pyr) bonds are slightly shorter, while Ga−
N(en) bonds are longer. The evenly distributed array of bond
3
4
+
c
4
5
+
c
c
H L + H ⇆ H L
0.24
ND
5
6
lengths suggests an excellent fit of [hox]2 with the Ga cation
−
3+
a
From ref 40, 50 v/v % aqueous dioxane solution, I = 0.1 M KCl.
b
2−
and a stability possibly comparable to that of [dedpa] (for
further structural data see the Supporting Information).+
Charges are omitted for clarity, ND = not determined. This work, I =
c
0
.16 M NaCl. This work, not evaluated at constant I = 0.16 M NaCl.
The coordination geometries of the [Ga(dedpa)] and
+
[
Ga(hox)] cations in aqueous solution were simulated using
H hox in its neutral form is denoted as H L, whereas its fully
protonated form in very acidic solution is H L . The latter
2
2
4
+
density functional theory (DFT); Figure 2a,b. The calculated
bond parameters of these two cations are summarized in
Tables S5 and S6 and compared with their solid-state
structures. Similar geometries and bond lengths are observed
in the simulated solution structures of both cations. The
corresponding molecular electrostatic potential mapping
6
possesses six potential protonation sites: two phenolate oxygen
atoms, two pyridyl nitrogen atoms, and the two secondary
amine nitrogen atoms on the en backbone. Hata and Uno
determined and assigned the first four protonation constants of
H hox by pH-potentiometric titrations in a H O−dioxane
2
2
(
[
(
MEP) of these geometries is shown in Figure 2c,d. In the
Ga(hox)] cation, less prominent electronegative potential
solvent mixture (50% v/v, μ = 0.1 M KCl, 25 °C) due to its
+
40,41
insufficient solubility in water.
In this work, we were able
indicated by red areas) is observed, which could result in a+
to analyze all six protonation events in water by exhaustive UV
spectrophotometric titrations, taking advantage of the high and
different molar absorptivities of each protonated species of
higher stability and a slower rate of hydrolysis of [Ga(hox)]
+
compared to that of [Ga(dedpa)] under acidic conditions.
Moreover, a more evenly distributed surface charge would
H hox. These titrations were performed from very acidic
solutions until pH 11.80, showing well-defined isosbestic
points (Figure S1a−f) indicative of consecutive deprotonation
2
+
translate into a higher lipophilicity for [Ga(hox)] versus
+
[
Ga(dedpa)] .
C
DOI: 10.1021/acs.inorgchem.8b01208
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Inorganic Chemistry
Article
steps of the ligand. Seven absorbing species were identified
H hox mixtures and the great stability of the metal complex;
2
(
Figure S2); six protonation constants were determined using
UV spectrophotometry was applied to individual samples
following the spectral changes on the ligand absorption bands
in the pH range from 0.19 to 11 (Figure S1). Samples
containing the complex and different amounts of standardized
HCl or NaOH were prepared, and because of the high stability
of the complex even at very acidic pH, the samples were
prepared and measured after 24 h, when equilibrium was
achieved (see acid-assisted dissociation study in Supporting
Information, Figure S4). The spectra of the Ga-hox complex
collected from pH 0.19 to pH 0.95 showed identical features to
those of the free ligand at the same pH values (Figure S3)
47
the HypSpec2014 program and summarized in Table 2. The
distribution diagram is presented in Figure 3a.
4
+
3+
suggesting the presence of the H L and H5L species that
6
2
+
deprotonate to the H4L species (λmax = 245 nm and λmax
=
3
08 nm) with two well-defined isosbestic points at λ = 249 nm
and λ = 346 nm, respectively (Figures S1a,b and S3a,b). There
is no complex formation evidence until pH > 1, when a new
band appears at λ = 368 nm as well as two isosbestic points at
λ = 251 and 335 nm and the shift of the band of the free ligand
at λ = 243 nm to lower energies at 260 nm due to the complex
formation (Figure 3c,d). There is no further transformation
along the pH range from 2 to 10.5. These data, together with
the molar absorption coefficients of the different absorbing
species of the free ligand (Figure S2), allowed the
determination of the stability constant of the Ga-hox complex
Figure 3. (a) Distribution diagram of H hox calculated using the
2
protonation constants in Table S1, at ligand concentration of 2.72 ×
47
−
5
3+
using the HypSpec program (Table 3).
1
[
0
M. (b) Distribution diagram of the Ga-hox system, [Ga]
=
−
5
H hox] = 2.82 × 10 M; 25 °C; I = 0.16 M NaCl. (c, d)
2
Table 3. Formation Constants of Ga3+ Complexes and pM
Values
a
Representative spectra in the batch UV spectrophotometric titration
3+
of the Ga -H hox system, 1:1 metal-to-ligand molar ratio, [H hox] =
2
2
.82 × 10− M, (l = 1 cm) at 0.16 M NaCl and 25 °C.
5
2
log K
log KML
34.35(1)
pGa
28.3
27.4
b
H hox
2
From the analysis of the spectra of H hox solutions between
2
c
H dedpa
28.11(8)
13.13(8)
*36.41(1)
30.98
2
pH 0−3.39 (Figure S1a,b), the ligand at its fully protonated
d
4
+
oxine (KP46)
form H L presents similar spectroscopic features to those of
6
+
48
21
27.9
fully protonated oxine (H L ) reported by both Choppin
and Enyedy, with almost doubled molar absorptivities for
2
ox
e
3
9
NOTA
DOTA
f
,
g
f
g
j
21.33; 26.05
18.5; 19.50
23.1
λmax = 260 and 378 nm consistent with two protonated
h
+
4+
TRAP
HBED
26.24
37.73; 39.57
quinolinium-NH chromophores (Table S1). The H L
6
i
,
j
i
i
3+
27.7; 29.4
species transforms into the H5L species with the appearance
of isosbestic points at 249 and 346 nm, which have been
reported also for the deprotonation of H L (Table S1). The
molar absorptivities for the H L species are of the same order
of magnitude as those of the fully protonated oxine (H L ),
k
DFO
28.65
21.2
a
3+
x−
+
Calculated at specific conditions ([Ga ] = 1 μM, [L ] = 10 μM,
2
ox
b
c
3+
pH 7.4 and 25 °C). This work (0.16 M NaCl at 25 °C). From ref
5
d
+
27 (0.16 M NaCl at 25 °C). From ref 39 (*) log K Ga(L ) (0.20 M
ox
3
2
ox
e
f
+
KCl at 25 °C). From ref 49 (0.10 M KCl at 25 °C). From ref 50 (0.1
consistent with one protonated quinolinium-NH . This allows
g h
M KCl at 25 °C). From ref 9 (0.1 M (NMe )Cl at 25 °C). From ref
4
for the assignment of the two most acidic pK values to the
i
j
k
a
1
8. From ref 25. From ref 51. From ref 52.
dissociation of the protons from the two quinoline nitrogen
atoms (pK = 0.24(8) and pK = 0.64(6)). In addition, the
1
2
−
2−
spectra of the species HL and L in most basic conditions
show spectroscopic evolution similar to that observed for the
quinolinate species of oxine (Lox−), with the appearance of
isosbestic points at 249 and 320 nm (Figure S1e,f and Table
In Figure 3b is shown the speciation plot of the Ga-hox
system calculated from the stability constants from Table 3.
The absorption band with maxima at 368 nm and ε = 4468
−
1
−1
+
M
cm in the [Ga(hox)] complex presents similarities with
3
9,48
39
S1).
This indicates the deprotonation of phenol−OH
that reported by Enyedy for the Ga(L ) species in water at I
ox 3
groups in both steps (pK = 9.81(1) and pK = 10.88(1)). The
equilibria involving the species H L , H L , and H L show
= 0.20 M KCl and 25 °C. The molar absorptivity Ga(L ) (ε
5
6
ox 3
3+
2+
−1
−1
= 6627 M cm , calculated from their spectra) is 1.5 times
the obtained value for [Ga(hox)] , which agrees with our
4
3
2
+
much smaller spectroscopic differences than those for the
previously mentioned processes (Figures S1c,d and S2). These
species present molar absorptivities doubled versus the neutral
HLox species (Table S1), which accounts for the loss of the
protons from the secondary amine nitrogen atoms on the
backbone (pK = 6.06(2) and pK = 8.39(1)).
results considering that, in H hox, there are two hydroxyquino-
2
line chromophores.
The high log KML = 34.35(1) value of the [Ga(hox)] ion
+
characterizes H hox as a very high stability chelator indeed for
2
3
+
Ga . Even more interesting are the conditional stability
constants or pM values (defined as −log[Mfree] at [L] = 10 μM
and [M] = 1 μM at pH = 7.4), which predict the stability of
the complexes in vivo and allow for the most suitable
3
4
Determination of the complex formation constant of
Ga(hox)] by direct pH-potentiometric method was not
+
[
possible due to both the insufficient solubility of the Ga−
D
DOI: 10.1021/acs.inorgchem.8b01208
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Inorganic Chemistry
Article
6
8
+
comparison of the relative ability of different ligands with
plasma competition experiment was performed. [ Ga(hox)]
53,54
different basicities to sequester a specific metal ion.
In
was incubated with mouse plasma for 5, 15, 30, and 60 min at
37 °C and analyzed by radio high-performance liquid
chromatography (HPLC). As shown in (Table 4), the complex
was completely intact (99+%) at all time points, confirming its
excellent in vitro stability of a whole half-life.
Table 3 are summarized the stability constants and calculated
68
pM values for some of the most important Ga chelators. The
6
8
Ga-hox pM value of 28.3, among the current reported Ga
chelator pM values, falls between the two literature-reported
values for HBED ligand, which has an even higher pM value
3
+
for Ga . This fact, together with the presence in solution of a
Table 4. Mouse Plasma Stability of 68Ga-hox
+
single species ([Ga(hox)] ) in the pH range of 1−11 and
5
min
15 min
>99%
30 min
>99%
1 h
considering that chelators such as DOTA, TRAP, the
39
55
68Ga-hox
antitumor Ga(L ) (KP46), or NOTA start to hydrolyze
>99%
>99%
ox 3
in the pH range of 7−9, represents a distinct advantage for
H hox and strongly suggests H hox as a promising ligand for
Dynamic PET/CT Imaging. The high in vitro stability,
2
2
gallium radiopharmaceutical compounds in a toolkit applica-
tion.
together with the high thermodynamic stability shown by
[Ga(hox)] , suggests high in vivo stability. A dynamic PET/
+
68
Ga Labeling Experiments. Encouraged by these most
CT imaging study in mice was therefore used to investigate the
6
8
68
+
promising solution studies, Ga labeling studies were
performed to investigate the coordination kinetics and
radiolabeling efficiency, crucial properties for an ideal PET
in vivo stability and biodistribution of the [ Ga(hox)] cation.
As shown in Figure 5, dynamic PET/computed tomography
68
radiopharmaceutical based on Ga. These studies show that
68
H hox coordinates Ga quantitatively within 5 min at room
2
temperature (faster than do H dedpa, NOTA, and DOTA).
2
DOTA, however, requires a high temperature (≥90 °C) for
quantitative yields and is thus incompatible with the labeling of
5
6
thermally sensitive biomolecules. In our experiments,
quantitative conversion (99% purity) was achieved with ligand
concentrations as low as 1 × 10− M (Figure 4), and the molar
7
Figure 5. (a) PET/CT dynamic imaging and (b) biodistribution of
6
8
+
[
Ga(hox)] in male (NRG) mice during (b) 0−3 min and (c) 3−53
min.
Figure 4. HPLC traces of radiation and UV absorption of the
CT) imaging showed no leakage of free 68Ga, which would
58
(
68
+
−7
+
mixtures of [ Ga(hox)] (CL = 1 × 10 M) and [Ga(hox)]
accumulate significantly in bone. There is no accumulation in
muscle as well. The complex showed quick heart uptake in the
first 2 min followed by fast clearance via both hepatobiliary
(liver, then gut) and renal (kidney, then bladder) pathways,
which could be explained by its small size and log D value
+
−5
nonradioactive complex ([Ga(hox)] = 5 × 10 M).
activity obtained was as high as 11 ± 1 mCi/nmol (407 ± 3.7
MBq/nmol) without any purification. The molar activity for
H hox is even higher than that of H dedpa (9.8 ± 1 mCi/
7
.4
+
2
2
(−0.47 ± 0.01). Even though the “naked” [Ga(hox)] is still a
hydrophilic complex based on its negative log D value, the
increased lipophilicity compared with most of the high polar
multiarmed carboxylate-based chelators, such as NOTA and
nmol), which was reported to be the highest obtained of all
6
8
12,27,57
Ga chelators with neither heating nor purification.
The
68
+
log D7.4 measurements showed [ Ga(hox)] was still a
reasonably hydrophilic complex, with an average log D7.4 of
29
DOTA, may elicit higher liver uptake. However, recent
studies with PSMA showed that the increased lipophilicity of
−
0.47 ± 0.01 (n = 4), even though it was more lipophilic than
6
8
NOTA, DOTA, and H dedpa.
the Ga-PSMA tracer using a more lipophilic HBED-CC
chelator translated into reduced unspecific binding and
increased specific tumor cell uptake and imaging quality
2
All of these chemistry advantages characterize H hox as a
2
superbly promising ligand for one-step kit-based labeling and
led to in vitro and in vivo experiments. Knowing that a suitably
dramatically, compared with the conjugate using the hydro-
2
7,35
23,59
substituted ethylenediamine precursor is available
to form
philic DOTA chelator.
In research reported recently, Yoo
an easily functionalizable (hox)2 ligand gave even stronger
−
and co-workers conjugated five different chelators with the
same RGD peptide and investigated the effect of lipophilicity
of the chelators on the biological behavior of the
impetus to these studies.
Mouse Plasma Competition Experiments. To inves-
tigate the in vitro stability of the 68Ga-hox system, a mouse
60
bioconjugates. Their research also revealed that bioconju-
E
DOI: 10.1021/acs.inorgchem.8b01208
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
gates with more lipophilic chelators have higher tumor uptake
as well as tumor/organ ratios, even though the liver uptake was
increased versus those bioconjugates with polar hydrophilic
chelators. Another pertinent example comes from our own
metal ion complexation by monitoring fluorescence changes
and concomitant dissociation of metal ion in stability studies of
bifunctional chelators and could therefore provide useful
information without the need of radioisotope experiments.
Second, the intrinsic fluorescence could enable a bimodal
tracer and direct fluorescence imaging of labeled bioconjugates
without an extraneous fluorescence tag that could alter
biological behavior by mutual disturbance of the two moieties
and/or affect the pharmacokinetics of the radiopharmaceutical.
One important advantage of a bimodal agent is that it can
combine complementary information obtained from separate
experiments to obtain a comprehensive synergistic analysis.
For example, in a study of somatostatin receptor imaging
agent, the unexpected results from in vivo PET/single photon
emission computed tomography (SPECT) imaging was finally
explained by optical fluorescence cellular imaging study of
endocytotic uptake, benefiting from its high spatial resolution,
1
11
work,
In-octapa-trastuzumab using a more lipophilic
picolinate-based chelator showed a markedly higher tumor
1
11
uptake than did In-DOTA-trastuzumab, which suggests that,
even for large biovectors, the effect of chelating moieties on the
61
pharmacokinetic properties may not be negligible. Therefore,
we posit that H hox is a good complementary choice for the
2
currently used chelator library in tuning the pharmacokinetics
of the bioconjugates, especially for the small highly polar
targeting vectors like the Glu-urea-Lys PSMA inhibitors.
The quick initial heart uptake also suggests that H hox could
2
be a useful scaffold in designing lipophilic cations for heart
imaging. The log P values between 0.8 and 1.2 have been
hypothesized to be optimal for good heart imaging contrast
68
6
2,63
which cannot obtained by just PET/SPECT imaging. For
clinical use, fluorescence techniques could also be useful
through endoscopy or improve the surgical excision in
tracers;
however, all the chelators currently used are too
hydrophilic for that application. We tried to design a series of
lipophilic cations based on H dedpa scaffold, but it is hard to
2
6
9
fluorescence directed surgery. Most of the previously
increase the log P value to the targeted region, without
34,38
reported bimodal (optical imaging and PET/SPECT) agents
increasing the molecular size too much.
Radiotracers with
70−75
require one fluorophore and one radioactive moiety.
The
appropriate log P have also been reported using a Schiff base
ligand; however, complex stability was sacrificed, because the
extraneous fluorescence tag, however, may affect the metal
74
6
4−67
sequestering capacity of the chelate moiety or require an
70,75
Schiff base complex was not metabolically stable.
extra spot for bioconjugation on the biovectors
and may
Therefore, we think, with this novel lipophilic H hox scaffold,
2
change the pharmacokinetics of the whole tracer as well.
a simple modification on the aromatic ring will increase the
6
8
Therefore, H hox is a significant discovery to obviate this dual-
lipophilicity and log P value and would provide novel Ga-
2
modality-dual-probe problem.
based candidates for heart-imaging applications.
+
To prove this concept, we investigated the subcellular
[
Ga(hox)] Fluorescence and Cell-Imaging Studies.
+
distribution and stability of [Ga(hox)] in living HeLa cells
H hox shows chelation-enhanced fluorescence properties. As
2
using fluorescence microscopy. HeLa cells were incubated with
shown in Figure 6, the peak emission wavelength of H hox in
2
1
50 μM [Ga(hox)][ClO ] for 2 or 24 h. Bright-field images of
4
phosphate-buffered saline (PBS) buffer (pH = 7.4) was ∼460
3+
treated cells (Figure S13) taken prior to fluorescence imaging
verified the cells as viable. The morphology of HeLa cells
appeared normal and suggested a low cellular toxicity of
nm and shifted to 560 nm once complexed with Ga , with a
fourfold increase in intensity. This property of H hox could
2
encompass two major advantages over the other reported
chelators. First, it could significantly inform nonradioactive
[
5
Ga(hox)][ClO ]. The fluorescence imaging was taken with a
20 nm emission filter, which allowed the detection of
4
fluorescence at wavelengths greater than 520 nm (Figure 6).
The complex was found to accumulate in cytoplasm, as Enyedy
39
et al. noted for the anticancer Ga(L ) complex. No obvious
ox 3
decomposition of the [Ga(hox)][ClO ] complex was observed
4
within the 24 h cellular environment, as the fluorescence
intensity was constant, and the free ligand would otherwise
exhibit markedly lower fluorescence intensity at wavelengths
above 520 nm (Figure 6).
This proof-of-concept study showed that this chelation-
enhanced fluorescence property could be used directly in
intracellular distribution and stability studies, which has never
been reported before with any other ligand. The intracellular
distribution could provide important preparatory information
for many other studies; for instance, Auger electron-based
therapy requires localization of the radionuclides in the nucleus
or hypoxia imaging tracers, which could be also evaluated using
fluorescent imaging with a multicellular spheroid tumor model
74
before radioactive in vivo study.
CONCLUSIONS
■
3
+
In summary, H hox, as a next-generation dual-channel acyclic
2
Figure 6. (upper) Fluorescence spectra of H hox and its Ga
2
3+
+
chelating ligand for Ga complexation, displays an unprece-
complex in PBS (λ = 365 nm, [H hox] = [Ga(hox)] = 1.7 ×
exc
2
−
5
dented array of properties, surpassing any ligand currently
1
0
M). (lower) Time-dependent fluorescence microscopy images
from HeLa cells treated with 150 μM [Ga(hox)] [ClO ]. The scale is
used. The synthesis of H hox is easier and more straightfor-
ward than that for any previously reported analogous chelator.
2
4
2
0 μm.
F
DOI: 10.1021/acs.inorgchem.8b01208
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
The higher thermodynamic stability of the [Ga(hox)]+
was added after the mixture cooled, and the mixture was filtered. The
filtrate was evaporated to solid crude and was purified by silica-gel
column chromatography (hexane/ethyl acetate, 10:90 to 50:50, v/v)
complex (log K = 34.4) compared to that of most relevant
ML
3+
Ga chelators and, most importantly, the largest pM value of
8.3 among those ligands proves the affinity of H hox toward
to obtain pure yellow needle crystal product (25 mmol, 4.4 g). Yield =
2
2
1
3+
68
82%. H NMR (300 MHz, CDCl
) δ 10.21 (d, J = 0.9 Hz, 1H), 8.32
3
Ga . Moreover, H hox showed fast and quantitative Ga
2
(
(
1
dd, J = 8.5, 0.8 Hz, 1H), 8.15 (s, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.62
t, J = 8.0 Hz, 1H), 7.42 (dd, J = 8.3, 1.2 Hz, 1H), 7.28 (dd, J = 7.7,
radiolabeling at room temperature and low concentration (1 ×
−
7
1
0
M) yielding a remarkably high molar activity of 11 ± 1
13
.3 Hz, 1H). C NMR (75 MHz, CDCl ) δ 192.8, 153.1, 150.3,
3
mCi/nmol (407 ± 3.7 MBq/nmol) without any purification.
Most importantly, mouse serum stability experiments and
dynamic PET imaging studies have shown high in vitro and in
vivo stabilities that correlate with the high thermodynamic
stability found in the solution studies. [ Ga(hox)] is also
quickly cleared from the mouse via hepatobiliary and renal
138.0, 137.6, 131.1, 130.6, 118.2, 118.1, 111.4, 77.2.
H hox (2). 8-Hydroxyquinoline-2-carboxaldehyde (1) (11.6 mmol,
2
2 g) was dissolved in 50 mL of ethanol; ethylenediamine (5.8 mmol,
3
87 μL) dissolved in 5 mL of ethanol was added dropwise, and the
68
+
reaction mixture was stirred at 60 °C for 4 h. A light yellow
precipitation formed and was collected and resuspended in 50 mL of
ethanol. NaBH (5 equiv) was added in portions, and the reaction
4
pathways. The lipophilicity of H hox provides a choice
2
mixture was stirred at room temperature overnight. HCl (20 mL; 6
M) was added then and stirred for 4 h to quench the reaction. The
pH of the reaction mixture was then readjusted to neutral using
NaOH (2 M), and the white precipitation was filtered and dried as
crude product. The crude product was further washed with water and
methanol to obtain the pure product (5.2 mmol, 1.97 g). Yield = 87%.
complementary to the current library of chelators, to tune
the pharmacokinetics of bioconjugates and design lipophilic
tracers for heart imaging or cell labeling. The intrinsic
+
fluorescence of [Ga(hox)] imaged HeLa cancer cells showing
accumulation in the cytoplasm and suggests strongly that this
compound could serve in dual-channel (bimodal) imaging or
fluorescence-directed surgery. The fluorescence emission red
shift and intensity increase (chelation-enhanced fluorescence)
1
H NMR (300 MHz, MeOD) δ 8.21 (d, J = 8.5 Hz, 1H), 7.50−7.38
(
1
m, 2H), 7.36 (dd, J = 8.3, 1.6 Hz, 1H), 7.10 (dd, J = 7.3, 1.6 Hz,
1
3
H), 4.20 (s, 2H), 3.03 (s, 2H). C NMR (75 MHz, MeOD) δ
3+
157.4, 154.1, 139.2, 138.1, 129.5, 128.4, 122.0, 119.0, 112.3, 54.7,
9.0, 48.8. LSMS m/z = 375.2. The product was recrystallized using
upon the complexation of H hox with Ga could be directly
2
4
used to study the stability of a nonradioactive bioconjugate in
methanol to obtain colorless needle crystals. H hox 2HCl used for
3+
2
vitro. The high affinity of H hox for Ga , confirmed in
2
titration was synthesized by mixing a solution of H hox (3) in
2
solution as well as in vitro and in vivo, together with the fast
quantitative radiolabeling at low concentrations and mild
tetrahydrofuran (THF) and 6 M HCl and drying in vacuo to afford a
white powder. LR-ESI-MS calcd for C H N O : m/z = 375.2;
2
2
23
4
2
+
conditions encourage the use of H hox for development of a
found: 375.2 [M + H] . H hox·2HCl used for titration was
synthesized by mixing a solution of H hox (2) in THF and 6 M
2
2
2
convenient toolkit radiopharmaceutical compound. Current
HCl and drying in vacuo to afford a white powder. Elemental analysis:
H hox·2HCl·0.5 H O, calcd % for C 57.9, H 5.52, N 12.28; found: C
efforts are focused on bifunctional analogues of the H hox
2
scaffold.
2
2
58.24, H 5.30, N 11.90.
[
Ga(hox)][ClO ](4). H hox (40 mg, 0.107 mmol) was dissolved
4
2
EXPERIMENTAL SECTION
■
in acetonitrile. Ga(ClO
)
3
·6H O (55 mg, 0.116 mmol) was added,
4
2
Materials and Methods. All solvents and reagents were
purchased from commercial sources (TCI America, Sigma-Aldrich,
Fisher Scientific) and used as received unless otherwise indicated. The
analytical thin-layer chromatography (TLC) plates used were
and the pH was adjusted to ∼5 using 0.1 M NaOH. The reaction
mixture was stirred for 1 h at 50 °C, dichloromethane (DCM) was
added to extract the product, and a yellow crystal suitable for X-ray
diffraction formed from layer diffusion of diethyl ether into the DCM
1
1
aluminum-backed ultrapure silica gel 60 Å, 250 μm thickness; H
extraction layer. H NMR (300 MHz, MeOD) δ 8.81 (d, J = 8.5 Hz,
and 13C NMR spectra were recorded at ambient temperature on
1H), 7.87 (d, J = 8.5 Hz, 1H), 7.76−7.53 (m, 1H), 7.41 (d, J = 8.3
Hz, 1H), 7.00 (d, J = 7.7 Hz, 1H), 4.76 (d, J = 17.8 Hz, 1H), 4.38 (d,
J = 17.9 Hz, 1H), 3.11 (d, J = 10.0 Hz, 1H), 2.63 (d, J = 9.9 Hz, 1H).
1
Bruker Avance 300 and Avance 400 spectrometers; the H NMR
spectra were calibrated against residual protio-solvent peak, and the
13
13
C NMR spectra were referenced to the deuterated solvent. Low-
C NMR (75 MHz, MeOD) δ 155.9, 150.2, 142.7, 134.9, 131.2,
+
resolution mass spectrometry was performed on a Waters ZG
spectrometer with an electrospray/chemical-ionization (ESCI)
source, and high-resolution electrospray ionization mass spectrometry
128.6, 120.2, 113.1, 112.7, 49.7. LR-ESI-MS for M of
6
9
C
H
22
22 GaN
O
2
: calcd m/z = 441.1; found 441.0. Elemental analysis
for [Ga(hox)][ClO ]·H O, calcd % C 47.22, H 3.96, N 10.01; found
C47.19, H 3.84, N 9.72.
X-ray Crystallography. Colorless tablet-shaped crystals of H hox
2
4
4
2
(
ESI-MS) was performed on a Micromass LCT time-of-flight (TOF)
instrument. Microanalyses for C, H, and N were performed on a
Carlo Erba Elemental Analyzer EA 1108. Purification and quality
were obtained by recrystallization from methanol. X-ray diffraction
data of a suitable crystal were collected using a Bruker APEX II area
detector diffractometer with Mo Kα radiation. The structure was
68
+
control of [ Ga(hox)] were performed on an Agilent HPLC system
equipped with a model 1200 quaternary pump, a model 1200 UV
absorbance detector, and a Bioscan NaI scintillation detector. The
radiodetector was connected to a Bioscan B-FC-1000 Flow-count
system, and the output from the Bioscan Flow-count system was fed
into an Agilent 35900E Interface, which converted the analog signal to
digital. The operation of the Agilent HPLC system was controlled
using the Agilent ChemStation software. The HPLC columns used
were a semipreparative column (Phenomenex C18, 5 μ, 250 × 10
mm) and an analytical column (Phenomenex C18, 5 μ, 250 × 4.6
solved in the monoclinic P2 /c space group. Yellow blade crystals of
1
[Ga(hox)][ClO ] suitable for X-ray diffraction were obtained layer
4
diffusion of diethyl ether into the DCM solution of the complex. X-ray
diffraction data of a suitable crystal was collected on a Bruker APEX
DUO diffractometer using Mo Kα radiation. The structure was solved
in the monoclinic C2/c space group. All non-hydrogen atoms were
refined anisotropically. All N−H hydrogen atoms were located in
difference maps and refined isotropically. All other hydrogen atoms
were placed in calculated positions. Further structural refinement
details and the .cif files are available in the Supporting Information.
DFT and TDDFT Calculations. All calculations were performed
using the Gaussian 09 package (Revision D.01). Full geometry
mm). The HPLC solvents were A: H O containing 0.1% trifluoro-
2
acetic acid (TFA) and B: CH CN containing 0.1% TFA. PET imaging
3
experiments were conducted using a Siemens Inveon microPET/CT
scanner.
Synthesis and Characterization. 8-Hydroxyquinoline-2-car-
boxaldehyde (1). A mixture of 8-hydroxyl-2-methylquinoline (31
mmol, 5 g) and selenium dioxide (35 mmol, 3.9 g) was stirred in 250
mL of 1,4-diethylene at 80 °C overnight. Diatomaceous earth (5 g)
+
optimizations of the [Ga(hox)] were performed with the CAM-
76
B3LYP hybrid exchange−correlation functional in aqueous solution
7
7
using the polarizable continuum model (PCE). Geometry
optimizations were performed using the 6-311+G(d,p) basis set on
G
DOI: 10.1021/acs.inorgchem.8b01208
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
first- and second-row elements and the Los Alamos effect core
potential (ECP) and valence basis set of double-ζ quality
phosphate buffer (pH 7.4). The mixture was vortexed for 1 min and
then centrifuged for 10 min. Samples of the octanol (1 mL) and buffer
78
(
LANL2DZ) on the Ga atom. The input coordinate of atoms was
(1 mL) layers were taken and counted. The log D7.4 value was
adapted from the crystal structure of the [Ga(hox)][ClO ] complex,
4
calculated using eq 1.
and no constraints on symmetry were imposed during the geometry
optimization. The resulting geometries showed no imaginary
frequencies and thus were confirmed to be minima on the potential
i counts in octanol phase y
j
z
j
z
log D7.4 = log j
z
10j
z
z
79
j
counts in buffer phase
energy surfaces. The PBE0 hybrid functional and the same basis set
and ECP was employed to simulate the UV−vis absorption features of
the fully optimized structure and generate its ground-state molecular
electrostatic potential (MEP) mapping.
k
{
(1)
6
8
Stability in Mouse Plasma. Purified Ga in 0.5 mL of water was
added into a 4 mL glass vial preloaded with 0.7 mL of 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (2 M,
Solution Thermodynamics. Protonation constants and metal
stability constants were calculated from UV spectrophotometric
titration data obtained using a Cary60 UV−vis spectrophotometer in
the spectral range of 200−450 nm. The path length was 1 cm for all
the samples. Individual samples of 5 mL containing the ligand
pH 5.0) and 25 nmol of H hox. The radiolabeling reaction was
2
performed under microwave heating for 1 min. The reaction mixture
was purified by HPLC using the semipreparative column eluted with
−
5
83/17 A/B (A: H
O containing 0.1% TFA, and B: CH CN
2 3
(
(
H hox, 2.72 × 10 M) or the corresponding gallium complex
2
+
−5
containing 0.1% TFA) at a flow rate of 4.5 mL/min. The retention
[Ga(hox)] , 2.82 × 10 M) in pure water were prepared by
6
8
+
time of [ Ga(hox)] was 13.8 min. The eluate fraction containing the
radiolabeled product was collected, diluted with water (50 mL), and
passed through a C18 Sep-Pak cartridge that was prewashed with
ethanol (10 mL) and water (10 mL). After the C18 Sep-Pak cartridge
adjusting the pH with different amounts of standardized HCl or
NaOH solutions, and NaCl was added to maintain a constant 0.16 M
ionic strength between the pH range of 0.8−11.85. A Ross
combination pH electrode was daily calibrated for hydrogen ion
37
68
concentrations using HCl as described before, and the results were
was washed with water (10 mL), the Ga-labeled product was eluted
80
analyzed by the Gran procedure. pH was measured in ligand and
metal−ligand samples between the pH range of 2.0−11.5.
off the cartridge with ethanol (0.4 mL), dried by helium, and
redissolved with saline (0.5 mL) for plasma stability and imaging
In the samples below pH 0.8, it was not possible to maintain
constant the ionic strength, since that depends on the HCl content,
68
+
studies. Aliquots (20 μL) of the [ Ga(hox)] were incubated with 80
μL of mouse plasma for 5, 15, 30, and 60 min at 37 °C. At the end of
each incubation period, samples were quenched with 100 μL of 70%
+
and the equilibrium H concentration was calculated from solution
stoichiometry, not measured with a glass electrode. For the solutions
0
81
CH CN and centrifuged for 20 min. The metabolites were measured
3
of high acidity, the correct acidity scale H was used. For the ligand
protonation equilibria study up to 2 min was attained at 25 °C to
reach the equilibrium before measuring the pH and the UV
absorption spectrum. For the samples containing the metal−ligand
complex, the measurements were performed only after 24 h when the
using a semipreparative HPLC system with the same HPLC
6
8
+
conditions as described for the purification of [ Ga(hox)] .
PET/CT Imaging Studies. PET/CT imaging studies were
conducted in accordance with the guidelines established by the
Canadian Council on Animal Care and approved by the Animal
Ethics Committee of the University of British Columbia. Male
equilibrium had been achieved. The protonation constants of H hox
2
and the Ga(III) complex stability constants were calculated from the
4
7
experimental data using the HypSpec2014 program. Proton
dissociation constants corresponding to hydrolysis of Ga(III) aqueous
tm1Mom
tm1Wjl
NOD.Cg-Rag1
Il2rg
/SzJ (NRG) mice were purchased
from in-house colonies at the Animal Research Centre, BC Cancer
Research Centre, Vancouver, Canada. PET/CT imaging experiments
were conducted using a Siemens Inveon microPET/CT scanner. Mice
were sedated with 2% isoflurane in oxygen inhalation and positioned
in the scanner. A baseline CT scan was obtained for localization and
attenuation correction before radiotracer injection, using 80 kV X-rays
at 500 mA, three sequential bed positions with 34% overlap, and 180°
continuous rotation. The mice were kept warm by a heating pad
during acquisition. The dynamic acquisition of 60 min was started at
82
ions included in the calculations were taken from Baes and Mesmer.
The species formed in the studied systems are characterized by the
general equilibrium: pM + qH + rL = M H L (charges omitted). For
p
q r
convention, a complex containing a metal ion M, proton H, and
ligand L has the general formula M H L . The stoichiometric indices p
p
q r
might also be 0 in the case of protonation equilibria, and negative
values of q refer to proton removal or hydroxide ion addition during
formation of the complex. The overall equilibrium constant for the
formation of the complexes M H L from its components is
p
q r
6
8
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 is defined as
the time of intravenous injection with ∼3.4−4.0 MBq of Ga−H hox.
2
The list mode data were rebinned into time intervals (12 × 10, 8 ×
60, 7 × 300, 1 × 900 s) to obtain tissue time−activity curves. Images
n+
n+
(
−log[M ] ) and is calculated at specific conditions ([M ] = 1
free
were reconstructed using iterative three-dimensional ordered subset
expectation maximization (OSEM3D, two iterations) using maximum
a priori with shifted poisson distribution (SP-MAP, 18 iterations).
Fluorescence Microscopy. Hela cells were purchased from the
American Type Culture Collection (ATCC). Cells were grown in
Eagle’s Minimal Essential Medium (MEM) supplemented with heat-
inactivated 10% fetal bovine serum, 1 mM sodium pyruvate, 4 mM L-
glutamine, and 1% nonessential amino acids in a humidified incubator
x−
μM, [L ] = 10 μM, pH 7.4 and 25 °C.
The acid-assisted dissociation kinetic study of the Ga(III)-hox
complex was performed by measuring the UV spectra in the same
experimental conditions as described above. Concentrated stand-
+
ardized HCl was added to a Ga-hox stock solution ([Ga(hox)] =
2
.82 × 10− M) to achieve pH 1. The reaction was followed by
5
registering the spectra at 15 min intervals at 25 °C.
68
+
68
[
Ga(hox)] Labeling Procedure. Ga was obtained from an
68
Eckert & Ziegler IGG100 Ga generator and was purified according
at 37 °C and 5% CO . Cells were seeded eight-well culture slips 24 h
2
83
to the previously published procedures using DGA resin column.
Radioactivity of [ Ga(hox)] was measured using a Capintec CRC
prior to treatment. The [Ga(hox)][ClO ] working solution for
4
68
+
fluorescence microscopy was prepared from a PBS stock solution. No
precipitation of the compound was observed in the working solution
under this condition. Cells were exposed to 150 uM [Ga(hox)]-
6
8
+
−
0
25R/W dose calibrator. [ Ga(hox)] was also obtained by adding
.2 mCi purified 68Ga to 200 mL of a 1 × 10 M solution of H hox
−7
2
in 0.1 M NaOAc solution (pH 8.5) and left for 5 min at RT. The
reaction progress was monitored by analytical HPLC eluted with 84/
[
ClO ] for 2 and 24 h and washed with PBS, and imaging was done
4
using a fluorescence microscope BX40, a U-MWU filter cube, an F-
View CCD camera (all Olympus), Cell-F fluorescence imaging
software (Olympus), and a 60× magnification oil immersion objective
lens.
1
6 phosphate buffer (pH 7.4)/CH CN at a flow rate of 2 mL/min.
3
6
8
+
The retention time of [ Ga(hox)] was 8.6 min.
Log D7.4 Measurements. Aliquots (2 μL) of the [ Ga(hox)]
were added to a vial containing 3 mL of octanol and 3 mL of 0.1 M
6
8
+
H
DOI: 10.1021/acs.inorgchem.8b01208
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(
7) Tsionou, M. I.; Knapp, C. E.; Foley, C. A.; Munteanu, C. R.;
ASSOCIATED CONTENT
Supporting Information
■
Cakebread, A.; Imberti, C.; Eykyn, T. R.; Young, J. D.; Paterson, B.
M.; Blower, P. J.; Ma, M. T. Comparison of macrocyclic and acyclic
chelators for gallium-68 radiolabelling. RSC Adv. 2017, 7, 49586.
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b01208.
(8) Bianchi, A.; Calabi, L.; Giorgi, C.; Losi, P.; Mariani, P.; Paoli, P.;
Rossi, P.; Valtancoli, B.; Virtuani, M. Thermodynamic and structural
properties of Gd3+ complexes with functionalized macrocyclic ligands
based upon 1,4,7,10-tetraazacyclododecane. J. Chem. Soc., Dalton
Trans. 2000, 697.
1
13
Detailed information on H NMR and C NMR spectra
of compounds, spectrophotometric measurements, X-ray
crystallography data (including tables of bond lengths
and angles), DFT-optimized geometries and bond
parameters, detailed HPLC radio-traces, and detailed
data of all biodistribution studies (PDF)
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9) Kubicek, V.; Havlickova, J.; Kotek, J.; Tircso, G.; Hermann, P.;
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AUTHOR INFORMATION
Corresponding Author
E-mail: orvig@chem.ubc.ca.
ORCID
Yang Cao: 0000-0001-8636-7919
Kuo-Shyan Lin: 0000-0002-0739-0780
Chris Orvig: 0000-0002-2830-5493
■
*
(
13) Ma, M. T.; Blower, P. J. In Metal Chelation in Medicine; The
Royal Society of Chemistry, 2017; p 260.
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temperature. Bioconjugate Chem. 2008, 19, 569.
(15) de Sa, A.; Matias, A. A.; Prata, M. I.; Geraldes, C. F.; Ferreira, P.
M.; Andre, J. P. Gallium labeled NOTA-based conjugates for peptide
Funding
receptor-mediated medical imaging. Bioorg. Med. Chem. Lett. 2010,
2
(
0, 7345.
Funding for this work was provided to C.O. by the Natural
Sciences and Engineering Research Council (NSERC) of
Canada as a Discovery Grant (RGPIN-42394-13), as well as by
NSERC (CHRP 493725-16) and the Canadian Institutes of
Health Research (CIHR, CPG-146482) as a Collaborative
Health Research Project.
16) Prata, M. I.; Santos, A. C.; Geraldes, C. F.; de Lima, J. J.
Structural and in vivo studies of metal chelates of Ga(III) relevant to
biomedical imaging. J. Inorg. Biochem. 2000, 79, 359.
(
17) Simecek, J.; Schulz, M.; Notni, J.; Plutnar, J.; Kubicek, V.;
Havlickova, J.; Hermann, P. Complexation of metal ions with TRAP
1,4,7-triazacyclononane phosphinic acid) ligands and 1,4,7-triazacy-
(
Notes
clononane-1,4,7-triacetic acid: phosphinate-containing ligands as
unique chelators for trivalent gallium. Inorg. Chem. 2012, 51, 577.
The authors declare no competing financial interest.
(18) Notni, J.; Hermann, P.; Havlickova, J.; Kotek, J.; Kubicek, V.;
Plutnar, J.; Loktionova, N.; Riss, P. J.; Rosch, F.; Lukes, I. A
triazacyclononane-based bifunctional phosphinate ligand for the
preparation of multimeric 68Ga tracers for positron emission
tomography. Chem. - Eur. J. 2010, 16, 7174.
ACKNOWLEDGMENTS
M. V. Tran, J. Chen, and J. Cudmore are thanked for their
important assistance.
■
(
19) Waldron, B. P.; Parker, D.; Burchardt, C.; Yufit, D. S.; Zimny,
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