Synthesis and characterization of lipophilic cationic Ga(III) complexes based on the H2: CHX dedpa and H2dedpa ligands and their 67/68Ga radiolabeling studies

Article abstract of DOI:10.1039/c6ra24070d

⁶⁸Ga is an attractive isotope for incorporation into a positron-emission tomography (PET) imaging agent, and is finding use as an alternative generator-produced isotope to ^99mTc particularly in imaging of myocardial perfusion. We have synthesized six new chelating ligands based on our previously reported H₂dedpa and H₂CHXdedpa scaffolds (CHX = cyclohexyl, H₂dedpa = 1,2-[[carboxypyridin-2-yl]methylamino]ethane). These ligands are designed to incorporate several lipophilic appendages at the secondary nitrogens, and upon coordination to ⁶⁸Ga(iii) will form lipophilic, cationic complexes designed to mimic the properties of other clinically relevant myocardial perfusion imaging agents. The non-radioactive Ga(iii) complexes were prepared and characterized by NMR spectroscopy; each ligand retained its predicted hexadentate N₄O₂ binding to Ga(iii). The radiolabeling properties of the six ligands were assessed using the longer-lived ⁶⁸Ga surrogate, ⁶⁷Ga. The absence of 'free' uncomplexed ⁶⁷Ga in the HPLC radio-chromatograms indicated >99% radiochemical yields (10 minutes at ambient temperature, ligand concentrations of 10^-4 M). However, the N,N′-benzyl functionalized derivatives displayed multiple peaks corresponding to the presence of additional ⁶⁷Ga-complexes which complicated further study. Selected ⁶⁷Ga-CHXdedpa complexes were tested for in vitro stability against the metal-binding protein apo-transferrin, and were found to be sufficiently stable (>80%) in a 2 h challenge assay, suggesting that alternative N,N′-alkylated derivatives which introduce more lipophilic character will be of interest in future studies.

Full text of DOI:10.1039/c6ra24070d

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Synthesis and characterization of lipophilic cationic
Ga(III) complexes based on the H2CHXdedpa and
H2dedpa ligands and their 67/68Ga radiolabeling
studies†
Cite this: RSC Adv., 2016, 6, 103763
C. F. Ramogida,ab D. Schindler,a C. Schneider,a Y. L. K. Tan,a S. Huh,a C. L. Ferreira,c
b
a
*
M. J. Adam and C. Orvig
68Ga is an attractive isotope for incorporation into a positron-emission tomography (PET) imaging agent,
and is finding use as an alternative generator-produced isotope to 99mTc particularly in imaging of
myocardial perfusion. We have synthesized six new chelating ligands based on our previously reported
H2dedpa and H2CHXdedpa scaffolds (CHX
¼
cyclohexyl, H2dedpa
¼
1,2-[[carboxypyridin-2-yl]
methylamino]ethane). These ligands are designed to incorporate several lipophilic appendages at the
secondary nitrogens, and upon coordination to 68Ga(III) will form lipophilic, cationic complexes designed
to mimic the properties of other clinically relevant myocardial perfusion imaging agents. The non-
radioactive Ga(III) complexes were prepared and characterized by NMR spectroscopy; each ligand
retained its predicted hexadentate N4O2 binding to Ga(III). The radiolabeling properties of the six ligands
were assessed using the longer-lived 68Ga surrogate, 67Ga. The absence of ‘free’ uncomplexed 67Ga in
the HPLC radio-chromatograms indicated >99% radiochemical yields (10 minutes at ambient
temperature, ligand concentrations of 10ꢀ4 M). However, the N,N0-benzyl functionalized derivatives
displayed multiple peaks corresponding to the presence of additional 67Ga-complexes which
complicated further study. Selected 67Ga-CHXdedpa complexes were tested for in vitro stability against
the metal-binding protein apo-transferrin, and were found to be sufficiently stable (>80%) in a 2 h
challenge assay, suggesting that alternative N,N0-alkylated derivatives which introduce more lipophilic
character will be of interest in future studies.
Received 28th September 2016
Accepted 19th October 2016
DOI: 10.1039/c6ra24070d
www.rsc.org/advances
MPI is dominated by the use of 99mTc labelled agents for
single-photon emission computed tomography (SPECT)
imaging. The FDA approved radiopharmaceuticals 99mTc-
Introduction
Myocardial perfusion imaging (MPI) is a common and powerful
technique used to illustrate the function of the heart muscle. Its
main application is in diagnosis of coronary artery disease (CAD)
and it is of great importance for early detection of heart disease, one
of the leading causes of death in the western world.1 The aim of
MPI is to visualise the perfusion of the heart muscle; this blood ow
analysis relies on the ensuing accumulation of a radiopharmaceu-
tical in the heart. Areas with good blood circulation will show
increased radionuclide uptake compared to areas with poor ow or
damaged tissue, thus dening the extent and severity of disease.1–4
sestamibi (Cardiolite), 99mTc-tetrofosmin (Myoview), and
99mTc-teboroxime (Fig. 1) are among the most popular agents
used clinically. It is believed that the elevated myocardial
accumulation of these 99mTc tracers is rooted in the physical
properties of the complexes: these are highly lipophilic and
monocationic or neutral charged complexes which contain at
least two ether-like linkages.4,5 Limitations such as low rst-pass
extraction and high liver uptake of such agents have sustained
interest in developing new radiopharmaceuticals for MPI. The
ideal perfusion radiotracer should have high heart uptake and
retention, with minimal liver and lung uptake to create diag-
nostically useful images.4
aMedicinal Inorganic Chemistry Group, Department of Chemistry, University of British
Columbia, 2036 Main Mall, Vancouver, British Columbia, Canada V6T 1Z1. E-mail:
orvig@chem.ubc.ca
The advantageous properties of the b+ emitter 68Ga (t1/2 ¼ 68
min) make it an ideal candidate for incorporation into an MPI
agent for positron-emission tomography (PET). A 68Ga-based
perfusion imaging agent would possess the specic advan-
tages of spatial resolution obtainable by PET compared to
SPECT, whilst relying on the convenience of a generator-
bLife Sciences Division, TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia,
Canada V6T 2A3
cNordion, 4004 Wesbrook Mall, Vancouver, British Columbia, Canada V6T 2A3
† Electronic supplementary information (ESI) available: 1H, 13C, 1H-1H COSY and
variable temperature NMR spectra of selected precursors, ligands, and complexes.
See DOI: 10.1039/c6ra24070d
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Fig.
2
Structures of novel lipophilic dedpa2ꢀ and CHXdedpa2ꢀ
analogues prepared herein (1–6).
Fig. 1 Commercially available 99mTc SPECT agents (top) and recently
investigated 68Ga PET agents (bottom) for myocardial perfusion
imaging.
a degree of added lipophilicity. The addition of ether groups
was found to reduce radiotracer liver uptake of cationic 99mTc
complexes resulting in improved target/background ratios.4 We
hope to take advantage of this theory with some of our deriva-
tives by incorporating such ether linkages in our ligands.
Indeed, many of the structural components that have been
added to this new class of compounds (1–6) have been strate-
gically chosen to mimic other 99mTc and 68Ga MPI agents under
investigation. The gallium coordination potential, labelling
properties, in vitro stability, and log P of the six chelating
ligands will be explored to assess their potential as myocardial
perfusion imaging agents.
produced isotope.6–8 Consequently, much effort has been made
towards development of new lipophilic, cationic Ga-complexes
with myocardial retention and low liver uptake.9–12 Speci-
cally, bis- and tris(salicylaldimine) chelators such as tris(4,6-
dimethoxysalicylaldimin-N,N0-bis(3-aminopropyl)-N,N0-ethyl-
enediamine) (BAPEN)9,13 and its derivatives10,11,14 (Fig. 1) showed
some promising properties for myocardial perfusion imaging
with 68Ga, but non-trivial labelling procedures have limited the
progression of these ligands towards the clinic.
Our recent reports of the promising acyclic hexadentate
67/68Ga chelator H2dedpa which forms a monocationic complex
with Ga(III) sparked interest in developing derivatives which
could potentially lead to compounds of increased and pro-
longed heart uptake.12,15 A small library of H2dedpa analogues
of varying lipophilicity which possess benzyl residues and
a variety of methoxy or ethoxy substituents was evaluated in
vivo.12 Despite modest biodistribution results showing initial
high uptake and slow clearance from the liver, two complexes
showed persistent heart uptake over the course of 2 h.12 These
conclusions have motivated us to develop a second class of
dedpa2ꢀ analogues which possess varying degrees of
lipophilicity.
Herein, we report ve new derivatives of the chiral acyclic
chelator H2CHXdedpa,16 and one new derivative of H2dedpa,
that have been functionalized to include varying lipophilic
appendages. Much like H2dedpa, the chiral chelator H2-
CHXdedpa forms 68/67Ga complexes of high thermodynamic
stability (log KML ¼ 27.61(8)), shows promising kinetic inertness
in vitro, and exhibits fast and mild 67/68Ga complexation kinetics
(radiochemical yields >99% in 10 minutes at RT at ligand
concentrations of 10ꢀ5 M), making H2CHXdedpa an ideal
candidate for further elaboration into a Ga(III) radiopharma-
ceutical.16,17 The six novel lipophilic analogues prepared here
contain either diethyl ether-like or benzyl linkages with varying
number of methoxy groups added onto the benzyl or 4-position
of the picolinate moieties (Fig. 2). Moreover, the cyclo-
hexanediamine backbone of CHXdedpa2ꢀ inherently introduces
Results and discussion
Ligand synthesis and characterization
The synthesis of pro-ligands 3–6 required the use of methyl 6-
(bromomethyl)-4-methoxypicolinate (10) which was prepared
using a previously reported synthesis.12 Using the methoxy-
modied bromopicolinate (10), the precursor 12 was prepared
using nosyl protection/deprotection chemistry (Scheme 1)
analogous to that used in the preparation of previous H2dedpa
and H2CHXdedpa analogues.16,18 The precursor 12 was nick-
named ‘Me2CHXdedpaOMe’ because it is similar to the previ-
ously synthesized analogue Me2CHXdedpa16,19 in which there
are no methoxy (OMe) groups appended to the 4-position of the
picolinate moieties.
A similar synthetic pathway was used to prepare ve ligands
(1–5) (Scheme 2). Preparation began with N,N0-alkylation of
Me2dedpa,15,18 Me2CHXdedpa,16 or Me2CHXdedpaOMe (12) with
a slight excess of the appropriate bromo-alkylating agent under
basic condition (K2CO3) to yield the methyl ester protected
precursors 13–17. For alkylation reactions with 1-bromo-2-
ethoxyethane (Br-ee), 2-ethoxyethane (ee) also replaced the
methyl protecting group of one or both of the carboxylic acid(s)
which resulted in a mixture of products. For precursors 14 and
15, separation and isolation of the over-alkylated products via
column chromatography was not possible, consequently 1H and
13C NMR spectra of pure compound were not collected. None-
theless, hydrolysis of all ester protected ligands was accom-
plished in the nal deprotection step using lithium hydroxide
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yield 18 which was subsequently used in an N,N0-alkylation
reaction with methyl 6-(bromomethyl)-4-methoxypicolinate
(10). Finally, the methyl-ester protected ligand 19 was depro-
tected using lithium hydroxide as was done for the previous
ligands to give H2CHXdedpaOMe-Bn3OMe (6).
1H NMR spectra of several pro-ligands at 25 ꢁC revealed very
broad and unresolvable peaks which precluded 13C NMR data
collection. It was hypothesized that intramolecular hydrogen-
bonding of the acidied ligand was the cause of such peak
broadening; however, NMR samples in deuterated water
adjusted to pD of >7.5 using NaOD resulted in only a slight
sharpening of peaks. Consequently, variable-temperature (VT)
NMR was used to collect 1H NMR spectra of the ligands at
Scheme 1 Synthesis of precursor 12, Me2CHXdedpaOMe. Reagents
and conditions: (i) methyl-6-(bromomethyl)-4-methoxypicolinate (2
equiv.), K2CO3, CH3CN, 65 ꢁC, 3 d; (ii) thiophenol (2.1 equiv.), K2CO3,
THF, RT, 3 d.
ꢁ
temperatures 25 to 75 C, and resulted in an apparent sharp-
ening of peaks with increasing temperatures (Fig. 3 and S1†);
13C NMR spectra were also obtained at the highest temperature.
Ga(III) complexation
Aer successful characterization of all six pro-ligands, prepa-
ration of metal complexes was attempted with non-radioactive
Ga(III). The dedpa2ꢀ and CHXdedpa2ꢀ based ligands 1–6 re-
ported herein were all able to complex Ga(III). In most cases,
upon addition of appropriate equivalency of Ga(ClO4)3 salt
solution to a solution of pro-ligand, precipitate formation was
observed; the precipitate was isolated and its identity as the Ga-
complex was conrmed by HR ESI-MS, and NMR spectroscopy.
Stacked 1H NMR spectra of two ligands and their corresponding
Ga-complexes are represented in Fig. 4 and S2.†
The diagnostic diastereotopic splitting of hydrogens upon
metal-complexation is seen in the NMR spectra of the dedpa2ꢀ
based ligand 1 (Fig. 4). The two singlets (labelled A and B),
assigned to the methylene hydrogens alpha to the pyridine ring
and the hydrogens on the ethylenediamine bridge, respectively,
of the ligand 1, become diastereotopic upon gallium complex-
ation and each singlet splits into two doublets (labelled a,a0 and
b,b0).
Scheme 2 Synthesis of pro-ligands H2dedpa-ee (1), H2CHXdedpa-ee
(2), H2CHXdedpaOMe-ee (3), H2CHXdedpaOMe-Bn (4), and H2-
CHXdedpaOMe-BnOMe (5). Reagents and conditions: (i) Na2CO3,
CH3CN, 75 ꢁC, 2 d; (ii) LiOH (5 equiv.), THF/H2O (3 : 1), RT, 30 min.
in order to yield all ve ligands 1–5 as pure compounds aer RP-
HPLC purication.
The commercial availability of an aldehyde starting material
(2,4,6-trimethoxybenzaldehyde) allowed ligand 6 to be prepared
in three synthetic steps, and avoided the need for nosyl
protection and deprotection chemistry which was utilized for
the preparation of ligands 1–5 through intermediate 12. Pro-
ligand 6 was synthesized in three steps (Scheme 3) through
imine formation followed by reductive amination of 2,4,6-tri-
methoxybenzaldehyde with (1R,2R)-(ꢀ)-cyclohexanediamine to
Diastereotopic splitting is visible in pro-ligands 2–6 due to
the inherently chiral nature of the CHXdedpa2ꢀ ligands.
1
Nonetheless, shis in the H resonances of the solution NMR
data (Fig. S2†) can be used to conrm successful Ga(III)
complexation.
67/68Ga radiochemistry and log P determination
With a half-life of 3.26 days, 67Ga is used as a longer-lived
surrogate for 68Ga complexes, and can be used in studies
requiring longer time points by monitoring its g-emission. 68Ga
was also used in the radiolabeling studies herein, when 67Ga
was not available. In all cases, the concentration of chelator
used for radiolabeling is in great excess relative to the radio-
isotope. Initial radiolabeling reactions were performed with
ligand concentrations of 10ꢀ4 M and 1 mCi of 67Ga in sodium
acetate buffer at pH 4.5, the reaction mixture was le for 10 min
at room temperature prior to injection into the HPLC.
Scheme
Reagents and conditions: (i) (a) ethanol, 0 ꢁC to RT, 18 h; (b) NaBH4,
ꢁC, 1.5 h; (ii) methyl-6-(bromomethyl)-4-methoxypicolinate (2.1
equiv.), Na2CO3, CH3CN, reflux to RT, 2 d. (iii) LiOH (5 equiv.), THF/H2O
(3 : 1), RT, 30 min.
3 Synthesis of pro-ligand H2CHXdedpaOMe-Bn3OMe (6).
The dedpa2ꢀ and both CHXdedpa2ꢀ ligands that possess 2-
ethoxyethane functionalities off the secondary amines (1, and
2–3, respectively) displayed one major sharp peak in the radio-
0
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Fig. 3 VT 1H NMR spectra of H2CHXdedpaOMe-N,N0-BnOMe (5) (400 MHz, D2O, 25–75 ꢁC).
1
Fig. 4 H NMR spectra at 25 ꢁC of (top) H2dedpa-N,N0-ee (1, 300 MHz, MeOD-d4) and (bottom) [Ga(1)]+ (400 MHz, DMSO-d6), highlighting
diastereotopic splitting of hydrogen resonances upon Ga-complexation. Peak A (top spectrum) splits into peaks a and a0 (bottom spectrum), and
peak B (top spectrum) splits into peaks b and b0 (bottom spectrum). *Residual solvent peak.
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chromatogram (99, 95, 99% RCY, respectively) assigned to the
67Ga-labelled complex. The remaining three CHXdedpaOMe
2ꢀ
ligands which possess benzyl functionalities attached to the
secondary amines with none, one, or three methoxy groups per
benzene ring (4, 5, and 6, respectively) display atypical 67Ga
labelling radio-traces. Despite the lack of free radioisotope
present in the HPLC radio-chromatogram (evinced by absence
of a peak at tR ¼ 2–3 min), three sharp peaks of varying intensity
are present over a span of about 3 min (Table 1 and Fig. 5). It is
possible that small impurities in the ligand solution are able to
complex with 67Ga more efficiently that the desired ligand,
resulting in multiple peaks in the radio-chromatogram. Ligand
impurities, even in small concentrations relative to the pure
ligand, can greatly affect labelling efficiency/yield since radio-
labeling conditions dictate that the concentration of the
chelator is in great excess compared to that of the radioisotope.
The peak with the latest retention time in the RP-HPLC radio-
trace is believed to be the desired 67Ga-complex, since the
chelating ligand will likely be the most lipophilic molecule in
the reaction mixture which will subsequently form the most
lipophilic 67Ga-complex. Further conrmation to correctly
identify the desired 67Ga-complex was performed by injecting
an aliquot of the pre-formed non-radioactive Ga-complex
([Ga(6)]+) into the HPLC under the same conditions as the
67Ga-complex and revealed one major peak in the UV-Vis
absorbance trace (tR ¼ 14.38 min, Fig. S28†) which matches
the latest peak in the radio-trace (tR ¼ 14.57 min, Fig. 5). Given
the shorter retention times of the two additional radio-peaks,
the impurities are believed to be the mono-alkylated and non-
alkylated by-products. The chelator purity was deemed suffi-
ciently pure post-purication by RP-HPLC by re-injection of
a small aliquot of chelator which resulted in one sharp peak and
by NMR spectroscopy, so it seemed surprising that the 67Ga
radio-traces of the above chelating ligands exhibited three
peaks of similar intensity. A second hypothesis is also plausible,
one in which multiple radiolabeling peaks arise from ligand
radiolysis. If ligand radiolysis is indeed occurring during the
course of the radiolabeling reaction, these ligands would be
deemed sufficiently unstable and unt for further testing in
vitro or in vivo. The steric bulk imposed by the benzyl groups on
pro-ligands 4, 5, and 6 may also contribute to the instability of
the chelators, resulting in ease of radiolysis or decomposition
Fig. 5 HPLC radio-chromatograms of 67Ga labelling reactions with
lipophilic dedpa2ꢀ or CHXdedpa2ꢀ chelators 1–6. * ¼ desired radio-
labelled product.
overtime. In an effort to distinguish whether or not the extra
67Ga-labelled products arise from ligand impurities or radiol-
ysis products, ligand 5 was radiolabelled with 1 mCi of 67Ga and
analysed by radio-HPLC at 10 minutes and again at 1 hour. It
was hypothesized that if the extra 67Ga-products at 10.91 and
11.76 min in the radio-trace were formed via ligand radiolysis,
the areas of these peaks would increase over time while the
assumed product peak at 13.97 min would decrease as the
radiative emissions of 67Ga induce more damage to the radio-
complex. However, the relative peak height and areas did not
change overtime (% RCYs were within ꢂ2% of each other,
Fig. S29†). Furthermore, ligand 6 was radiolabelled with 0.1 mCi
of 68Ga at constant ligand concentration (10ꢀ4 M) and analysed
via RP-HPLC aer 10 minutes at ambient temperature. Again,
the ratio (via % area) of the three 68Ga-labelled peaks in the
radio-trace did not change signicantly (23, 60, 16% compared
to 23, 54, and 21% for 67/68Ga-products from shortest to longest
retention time, Fig. S30†). We predicted that if indeed the extra
peaks at shorter retention were due to ligand radiolysis the
relative areas of impurity products would change compared to
the desired product peak between the [67Ga(6)]+ and [68Ga(6)]+
radiolabeling reaction, since radiolysis is dependent on factors
such as the time, amount, and type of radiation exposure; 67Ga,
a gamma and Auger electron emitter, would inict a different
destructive dose to the ligand compared to the primarily posi-
tron emitter 68Ga. The observation of little to no change in the
radio-traces of both the 67Ga- and 68Ga-labelled products of
ligand 6 further supports the hypothesis that ligand impurities
in the benzyl-alkylated ligands 4–6 have contributed to the
presence of multiple peaks in the radiolabeling reactions. The
steric bulk imposed by the benzene rings' close proximity to the
cyclohexane ring may be contributing to the instability of pro-
ligands 4–6, causing ligand decomposition via de-alkylation of
one or both of the secondary amines.
Table 1 Radiolabeling yields, corresponding HPLC radio-chromato-
gram retention times, and log P values for 67Ga-labelled dedpa2ꢀ and
CHXdedpa2ꢀ chelators 1–6
Complex
tR/min (RCY/%)
log P
[
[
[
[
[
[
67Ga(1)]+
67Ga(2)]+
67Ga(3)]+
67Ga(4)]+
67Ga(5)]+
67Ga(6)]+
8.85 (99)
11.50 (99)
12.68 (93)
ꢀ2.67
ꢀ1.76
ꢀ1.53
NDb
11.15a (20), 11.40a (27), 13.42 (32)
10.91a (23), 11.76a (53), 13.97 (24)
11.76a (23), 12.34a (54), 14.57 (21)
NDb
Determination of partition coefficients (log P) for myocardial
perfusion imaging agents is of particular importance because it
is believed that differences in heart uptake and liver clearance
are caused by the compound lipophilicity which affects the
NDb
Retention time of 67Ga-labelled by-products produced from ligand
a
b
impurities or ligand radiolysis. Not determined.
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ability to cross the plasma and mitochondrial membranes (the chelate complex. The in vitro stability of the pure 67Ga-
accepted mechanism of heart uptake).4 It has been hypothe- complex ([67Ga(CHXdedpaOMe-BnOMe)]+) may differ from the
sized that cationic 99mTc radiotracers should exhibit log P stability of the other side products in the mixture, such as the
values ranging from 0.5–1.2 in order to achieve optimal mono-alkylated ligand.
myocardium retention and fast liver clearance concurrently;4
therefore, we sought to nd a 68Ga-CHXdedpa complex which
Experimental
Materials and methods
exhibited a partition coefficient within this range.
Due to the presence of multiple species in the 67Ga-labeling
reactions of benzyl functionalized ligands 4–6, (vide supra) log P All solvents and reagents were purchased from commercial
values were not determined, since the value obtained through suppliers (Sigma Aldrich, TCI America, Fisher Scientic) and
experimental procedures would not accurately depict the lip- were used as received. Human apo-transferrin was purchased
ophilicity of the desired radiometal–ligand complex but instead from Sigma Aldrich. The analytical thin-layer chromatography
˚
would be inuenced by the mixture of the relatively hydrophilic (TLC) plates were aluminum-backed ultrapure silica gel 60 A,
67Ga by-products present in the reaction mixture. log P of the 250 mm thickness; the ash column silica gel (standard grade,
three 67Ga-ligands functionalized with ee groups (1–3) were 60 A, 40–63 mm) was provided by Silicycle. 1H and 13C NMR
˚
determined to be ꢀ2.67, ꢀ1.76, and ꢀ1.53, respectively. The spectra were recorded at 25 ꢁC unless otherwise noted on Bruker
structure of ligands 1 and 2 differ only by the presence on the AV300, AV400, or AV600 instruments; NMR spectra are
cyclohexane ring on 2, consequently by comparing directly the expressed on the d scale and referenced to residual solvent
log P values of [67Ga(1)]+ and [67Ga(2)]+ one can discern the peaks. Low-resolution mass spectrometry was performed using
lipophilic character caused by the ‘CHX’ backbone. The addi- a Waters ZG spectrometer with an ESCI electrospray/chemical-
tion of the cyclohexane ring to the ethylenediamine backbone ionization source, and high-resolution electrospray-ionization
resulted in a shi of 0.91 log P units between [67Ga(1)]+ and mass spectrometry (HR-ESI-MS) was performed on a Micro-
[67Ga(2)]+. Moreover, the added lipophilic character of [67Ga(3)]+ mass LCT time-of-ight instrument at the Department of
due to the addition of two extra methoxy groups off the 4- Chemistry, University of British Columbia. 67Ga-(chelate) apo-
position of the picolinate moiety are represented by a shi of transferrin stability experiments were analyzed using GE
0.23 log P units compared to [67Ga(2)]+ (in which there is no Healthcare Life Sciences PD-10 desalting columns (size exclu-
additional methoxy groups). The comparatively hydrophilic sion for MW < 5000 Da) and counted with a Capintec CRC 15R
nature of the three evaluated complexes renders their log P well counter. The HPLC system used for analysis and purica-
values well out of the optimal range predicted by Liu.4 None- tion of nonradioactive compounds consisted of a Waters 600
theless, valuable lessons on ligand design of lipophilic H2dedpa controller, Waters 2487 dual wavelength absorbance detector,
and H2CHXdedpa analogues have been drawn from the ndings and a Waters delta 600 pump. Phenomenex Synergi Hydro-RP
˚
of the structures (1–6) studied herein.
80 A columns (250 mm ꢃ 4.6 mm analytical or 250 mm ꢃ
21.2 mm semipreparative) were used for purication of several
of the deprotected ligands. Analysis of radiolabelled complexes
was carried out using a Phenomenex Synergi 4 m Hydro-RP 80A
analytical column (250 ꢃ 4.60 mm 4 mm) using either a Waters
Alliance HT 2795 separation module equipped with a Raytest
Gabi Star NaI (Tl) detector and a Waters 996 photodiode array
(PDA) or an Agilent HPLC system equipped with a model 1200
quaternary pump, a model 1200 UV absorbance detector (set at
250 nm), and a Raytest Gabi Star NaI(Tl) detector. 67GaCl3 was
cyclotron-produced and provided by Nordion as ꢄ0.1 M HCl
solution. 68Ga was obtained from an Eckert & Ziegler (Berlin,
Germany) IGG100 68Ge/68Ga generator and was puried
according to previously published procedures20 using a DGA
resin column.
Human apo-transferrin stability studies
Selected 67Ga-complexes were investigated for their stability in
a human apo-transferrin challenge. Compared to unaltered
[67Ga(dedpa)]+ which remained 99% intact aer 2 hours,15 the
new CHXdedpa2ꢀ derivatives with N,N-functionalization dis-
played moderately reduced stabilities of 81, 86, and 80% for
[67Ga(2)]+, [67Ga(3)]+, and [67Ga(5)]+ aer 2 hours, respectively
(Table 2). It should be noted that the [67Ga(5)]+ labelling reac-
tion used for the stability assay contained a mixture of labelled
products; as such the results of the transferrin challenge are
representative of the bulk and not the desired single 67Ga-
Precursors 7–10 (Scheme 4) were prepared using a modied
2
In vitro stability of selected 67Ga-labelled CHXdedpa2ꢀ
synthesis according to literature protocol of
analogue.21
a similar
Table
complexes (2, 3, 5) against human apo-transferrin (37 ꢁC, 2 h), with
stability shown as the percentage of intact 67Ga complex
Dimethyl-4-hydroxypyridine-2,6-dicarboxylate (7). Chelida-
mic acid (4.00 g, 19.9 mmol) was suspended in methanol (40
Complex
15 min (%)
1 h (%)
2 h (%)
ꢁ
mL) at ꢀ10 C. To this murky solution, thionyl chloride (11.4
[
[
[
67Ga(2)]+
67Ga(3)]+
67Ga(5)]+a
86.3
85.0
97.5
84.8
85.9
82.9
81.4
85.6
80.0
mL, 157 mmol, ꢄ8 equiv.) was added slowly. The solution,
which turned clear upon addition, was stirred and allowed to
warm to room temperature overnight. The reaction mixture was
then reuxed for 2 hours, subsequently cooled to room
temperature and concentrated in vacuo. The resulting solids
a
Multiple 67Ga products present in reaction mixture.
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Methyl-6-(bromomethyl)-4-methoxypicolinate (10). To a solu-
tion of 9 (0.74 g, 3.75 mmol) in chloroform (50 mL) at 0 ꢁC, PBr3
(0.42 mL, 4.50 mmol, 1.2 equiv.) was added. The reaction mixture
was allowed to warm to room temperature and stirred for 1.5
hours, while monitoring the reaction progress by TLC (Rf ¼ 0.63,
EtOAc). The mixture was subsequently quenched with cold
saturated K2CO3 (40 mL), transferred to a separatory funnel and
the organic layer was collected. The aqueous layer was then
washed with dichloromethane (3 ꢃ 50 mL). The organic fractions
were collected and dried over MgSO4. The organic solution was
concentrated in vacuo to yield the product as a pink solid (0.70 g,
71%). 1H NMR (300 MHz, CDCl3) d 7.60 (d, J ¼ 2.3 Hz, 1H), 7.17
(d, J ¼ 2.3 Hz, 1H), 4.59 (s, 2H), 4.01 (s, 3H), 3.93 (s, 3H). 13C NMR
(75 MHz, CDCl3) d 166.6, 164.6, 158.1, 148.5, 112.0, 110.3, 55.2,
52.4, 32.8.
Scheme 4 Synthesis of precursors 7–10. Reagents and conditions: (i)
SOCl2, CH3OH; (ii) methyl iodide, K2CO3, CH3CN, reflux; (iii) NaBH4,
CH3OH/CH2Cl2 (2 : 1), 0 ꢁC, 2 h; (iv) (1) PBr3, CHCl3, 0 ꢁC–RT, 1.5 h, (2)
Dimethyl-6,60-((((1R,2R)-cyclohexane-1,2-diyl)bis(((2-nitrophenyl)
sulfonyl)azanediyl))bis-(methylene))bis(4-methoxypicolinate) (11).
To a stirred solution of N,N0-((1R,2R)-cyclohexane-1,2-diyl)bis(2-
nitrobenzene-sulfonamide)16 (0.28 g, 0.58 mmol, 1 equiv.) and
K2CO3 (0.48 g, 3.6 mmol, 6 equiv.) in CH3CN (10 mL), methyl-6-
(bromomethyl)-4-methoxypicolinate (10) (0.31 g, 1.16 mmol, 2
equiv.) in CH3CN (5 mL) was added, and the reaction mixture
was stirred at reux for 20 hours. The excess inorganic salts
were subsequently removed by centrifugation (4000 rpm, 10
min) and ltration, and the ltrate was concentrated in vacuo.
The crude product was puried by column chromatography
(CombiFlash Rf automated column system; 24 g HP silica; A:
hexanes, B: ethyl acetate, 70% A to 100% B gradient) to yield the
product as a yellow solid (0.46 g, 94%). (TLC Rf (product) ¼ 0.67,
0
ꢁC, aq K2CO3.
were redissolved in hot ethanol, and cooled in freezer to
recrystallize. The recrystallized product was collected by
vacuum ltration, and the ltrate was concentrated to half-
volume, and the recrystallization procedure was repeated to
1
recover more solid product (2.597 g, 62%). H NMR (400 MHz,
MeOD) d 7.88 (s, 2H), 5.01 (s, 3H). 13C NMR (101 MHz, MeOD)
d 174.8, 161.5, 145.4, 117.7, 54.8. MS (ES+) m/z ¼ 212.3 [M + H]+.
Dimethyl-4-methoxypyridine-2,6-dicarboxylate (8). Compound
7 (1.941 g, 9.2 mmol) and potassium carbonate (3.9 g, 28.2 mmol,
ꢄ3 equiv.) were suspended in acetonitrile (50 mL). To this solu-
tion, methyl iodide (1.15 mL, 18.4 mmol, 2 equiv.) was added,
and the murky solution was stirred at reux overnight.
The resultant mixture was cooled to room temperature, ltered
by vacuum ltration to remove excess salts and the ltrate was
concentrated in vacuo. The crude solid was puried by column
chromatography (CombiFlash Rf automated column system; 80 g
HP silica; A: hexanes, B: ethyl acetate, 100% A to 100% B gradient)
1
100% EtOAc). H NMR (300 MHz, CDCl3) d 7.92 (d, J ¼ 7.7 Hz,
2H), 7.54 (m, 4H), 7.42 (m, 4H), 7.23 (m, 2H), 5.05 (d, J ¼
16.7 Hz, 2H), 4.61 (d, J ¼ 16.7 Hz, 2H), 4.34 (s, 2H), 3.90 (s, 6H),
3.86 (s, 6H), 2.21 (d, J ¼ 7.0 Hz, 2H), 1.71 (m, 2H), 1.42 (m, 2H).
13C NMR (75 MHz, CDCl3) d 166.8, 165.4, 158.5, 148.2, 147.7,
133.7, 133.0, 131.9, 131.3, 123.5, 111.5, 59.7, 55.8, 52.6, 50.2,
32.2, 25.2. MS (ES+) m/z ¼ 881.4 [M + K]+.
1
to yield the product as a white solid (1.384 g, 67%). H NMR
Dimethyl-6,60-((((1R,2R)-cyclohexane-1,2-diyl)bis(azanediyl))
bis(methylene))bis(4-methoxypicolinate) (12). To a solution of
dimethyl 11 (0.46 g, 0.54 mmol, 1 equiv.) and K2CO3 (0.90 g,
6.52 mmol, 12 equiv.) in tetrahydrofuran (20 mL), thiophenol
(116 mL, 1.14 mmol, 2.1 equiv.) was added and the mixture was
stirred at room temperature for 70 hours. The inorganic salts
were subsequently ltered out aer centrifugation (4000 rpm, 10
min) and the ltrate was concentrated in vacuo. The crude
product was puried by column chromatography (CombiFlash Rf
automated column system; 12 g HP silica; A: dichloromethane, B:
2% triethylamine in methanol, 100% A to 10% B gradient) to
yield the product as a yellow oil (0.25 g, 99%). 1H NMR (300 MHz,
CDCl3) d 7.40 (d, J ¼ 2.1 Hz, 2H), 7.10 (d, J ¼ 2.1 Hz, 2H), 3.99 (d, J
¼ 15.0 Hz, 2H), 3.83 (d, J ¼ 15.0 Hz, 2H), 3.81 (s, 6H), 3.72 (s, 6H),
3.01 (s, 2H), 2.23 (d, J ¼ 8.7 Hz, 2H), 2.01 (m, 2H), 1.58 (d, J ¼
(400 MHz, CDCl3) d 7.62 (d, J ¼ 4.9 Hz, 2H), 3.84 (s, 6H), 3.83 (s,
3H). 13C NMR (101 MHz, CDCl3) d 167.0, 164.4, 149.2, 113.4, 55.5,
52.5.
Methyl-6-(hydroxymethyl)-4-methoxypicolinate (9). Compound
8
(1.384 g, 6.15 mmol) was dissolved in methanol/
dichloromethane (2 : 1, 90 mL) and cooled to 0 ꢁC. Sodium
borohydride (0.233 g, 6.15 mmol) was added in small portions,
and the reaction mixture was stirred at 0 ꢁC for 2.5 hours. The
reaction mixture was quenched with water (50 mL), and the
organics were decanted and collected. The aqueous phase was
extracted with dichloromethane (8 ꢃ 50 mL), organics were
collected, dried over MgSO4 and all the organics were
concentrated in vacuo. The crude product was puried by
column chromatography (CombiFlash Rf automated column
system; 40 g HP silica; A: hexanes, B: ethyl acetate, 100% A to
100% B gradient) to yield the product as a white solid (0.871 g,
72%). 1H NMR (400 MHz, CDCl3) d 7.40 (d, J ¼ 2.3 Hz, 1H), 7.03
(d, J ¼ 2.2 Hz, 1H), 4.70 (s, 2H), 3.84 (s, 3H), 3.79 (s, 3H). 13C
NMR (101 MHz, CDCl3) d 167.2, 165.5, 163.0, 148.3, 110.4,
109.0, 64.7, 55.5, 52.8.
7.5 Hz, 2H), 1.07 (dd, J ¼ 17.8, 8.9 Hz, 2H), 0.96–0.86 (m, 2H). 13
C
NMR (75 MHz, CDCl3) d 166.9, 165.7, 163.0, 148.6, 110.9, 110.0,
61.2, 55.4, 52.7, 52.0, 31.4, 24.7. MS (ES+) m/z ¼ 473.4 [M + H]+.
Me2dedpa-N,N0-ee (13). To a solution of Me2dedpa15,18
(0.151 g, 0.42 mmol, 1 equiv.) and K2CO3 (0.349 g, 2.52 mmol, 6
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equiv.) in CH3CN (5 mL), 2-bromoethyl ethyl ether (119 mL, were added Na2CO3 (0.53 g, 5.00 mmol, ꢄ6 equiv.) and 1-
1.05 mmol, 2.5 equiv.) was added. The mixture was stirred for bromomethyl-3-methoxybenzene (243 mL, 1.7 mmol, 2 equiv.).
ꢁ
24 h at room temperature. Inorganic salts were ltered out by The mixture was stirred at 60 C overnight, aer which excess
centrifugation and ltration; the organic solution was concen- salts were removed by centrifugation (4000 rpm for 10 min) and
trated in vacuo. The product was puried by column chroma- ltration; the ltrate concentrated in vacuo. The crude product
tography (CombiFlash Rf automated column system; 12 g HP was puried by column chromatography (CombiFlash Rf auto-
silica; A: dichloromethane, B: methanol, 100% A to 5% B mated column system; 24 g HP silica; A: dichloromethane, B:
gradient) to yield pure product (0.159 g, 75%). 1H NMR (300 methanol, 100% A to 20% B gradient) to give the product as
MHz, CDCl3) d 7.94–7.86 (m, 2H), 7.75–7.65 (m, 4H), 3.92 (s, a yellow oil (0.48 g, 85%). HR-ESI-MS m/z for C40H49N4O8 (M +
4H), 3.90 (s, 6H), 3.42 (t, J ¼ 5.3 Hz, 4H), 3.32 (q, J ¼ 7.0 Hz, 4H), H)+ calcd (found): 713.3550 (713.3548) (ꢀ0.3 ppm).
2.74 (d, J ¼ 11.7 Hz, 8H), 1.05 (t, J ¼ 7.0 Hz, 6H). 13C NMR (75
(1R,2R)-N1,N2-Bis(2,4,6-trimethoxybenzyl)cyclohexane-1,2-
MHz, CDCl3) d 165.8, 147.0, 137.4, 126.3, 123.5, 68.5, 66.4, 60.8, diamine (18). To a solution of (1R,2R)-(ꢀ)-cyclohexanediamine
54.3, 53.0, 52.8, 15.1. MS (ES+) m/z ¼ 503.3 [M + H]+, 525.3 [M + (0.162 g, 1.42 mmol, 1 equiv.) in ethanol (8 mL) at 0 ꢁC, a solu-
Na]+.
tion of 2,4,6-trimethoxybenzaldehyde (0.557 g, 2.84 mmol, 2
Me2CHXdedpa-N,N0-ee (14). To a solution of Me2CHXdedpa16 equiv.) in ethanol/methanol (1 : 1, 20 mL) was added. The
(0.231 g, 0.56 mmol, 1 equiv.) dissolved in CH3CN (5 mL), 2- reaction mixture was stirred overnight and allowed to warm to
bromoethyl ethyl ether (133 mL, 1.18 mmol, 2.1 equiv.) and RT. Bis-imine formation was conrmed by ES-MS (m/z ¼ 471.4
Na2CO3 (0.356 g, 3.36 mmol, 6 equiv.) were added. The reaction [M + H]+), and the solution was again cooled to 0 ꢁC on ice.
mixture was stirred at 75 ꢁC for 48 h, subsequently cooled to RT Sodium borohydride (0.17 g, approx. 3 equiv.) was added to the
and the salts were removed by vacuum ltration; the ltrate was reaction mixture in small portions, and the mixture was stirred
concentrated in vacuo. The crude product was puried by for 1.5 hours. The mixture was then quenched with saturated
column chromatography (CombiFlash Rf automated column NH4Cl (20 mL), and extracted with dichloromethane (3 ꢃ 40
system; 24 g HP silica; A: dichloromethane, B: methanol, 100% mL). The organics were collected, dried over MgSO4 and
A to 10% B gradient) to yield the product as a uffy off-white concentrated in vacuo to yield the product as a faint yellow solid
solid (0.185 g) in a mixture also containing over-alkylated (0.675 g, >99%) which was used in the subsequent step without
1
products where 2-ethoxyethane is attached to the ester func- further purication. H NMR (400 MHz, MeOD) d 6.22 (s, 4H),
tion(s). The mixture could not be further separated. MS (ES+) m/ 4.10 (d, J ¼ 12.5 Hz, 2H), 3.82 (s, 6H), 3.79 (d, J ¼ 12.5 Hz, 2H),
z ¼ 557.4 [M + H]+.
3.71 (s, 12H), 2.48 (s, 2H), 2.24 (d, J ¼ 7.3 Hz, 2H), 1.81 (s, 2H),
Me2CHXdedpaOMe-N,N0-ee (15). To
a
solution of Me2- 1.30 (d, J ¼ 6.3 Hz, 4H). 13C NMR (75 MHz, MeOD) d 163.4,
CHXdedpaOMe (12) (79 mg, 0.17 mmol, 1 equiv.) in CH3CN (2 160.7, 105.0, 91.6, 61.1, 56.2, 56.0, 39.2, 29.9, 25.5. MS (ES+) m/z
mL), 1-bromo-2-ethoxyethane (66 mL, 0.58 mmol, 3.5 equiv.) and ¼ 475.4 [M + H]+.
Na2CO3 (106 mg, 1.00 mmol, 6 equiv.) were added. The reaction
Me2CHXdedpaOMe-N,N0-Bn3OMe (19). To
a solution 15
mixture was stirred at 80 ꢁC for 96 h. Excess salts were removed (0.276 g, 0.58 mmol, 1 equiv.) and methyl-6-(bromomethyl)-4-
by centrifugation (4000 rpm for 10 min) and the ltrate was methoxypicolinate (10) (0.322, 1.23 mmol, 2.1 equiv.) in
concentrated in vacuo. The crude product was puried by CH3CN (30 mL), Na2CO3 (0.355 g, 3.35 mmol, 5.6 equiv.) was
column chromatography (CombiFlash Rf automated column added, and the reaction mixture was stirred at reux for 6 hours
system; 24 g HP silica; A: DCM, B: methanol, 100% A to 20% B and subsequently at RT for 2 days. The excess salts were
gradient) and the product (yellowish oil (122 mg)) was obtained removed by vacuum ltration, and the ltrate was concentrated
as a mixture of product 6.7 and over-alkylated products where 2- in vacuo. The crude product was puried by column chroma-
ethoxyethane is attached to the ester function(s). The mixture tography (CombiFlash Rf automated column system; 24 g HP
could not be further separated. MS (ES+) m/z ¼ 639.6 [M + Na]+. silica; A: dichloromethane, B: methanol, 100% A to 10% B
Me2CHXdedpaOMe-N,N0-Bn (16). To a solution of Me2- gradient) to yield the product as a uffy white solid (0.382 g,
1
CHXdedpaOMe (12) (110 mg, 0.220 mmol) in CH3CN (2 mL), 79%). H NMR (400 MHz, CDCl3) d 7.89 (s, 2H), 7.59 (s, 2H),
bromobenzene (68 mL, 0.576 mmol, 2.5 equiv.) and Na2CO3 5.87–5.75 (m, 4H), 4.67 (d, J ¼ 14.2 Hz, 2H), 4.30 (d, J ¼ 13.8 Hz,
(0.15 g, 1.38 mmol, 6 equiv.) were added. The reaction mixture 2H), 4.10 (d, J ¼ 13.7 Hz, 2H), 4.00 (s, 6H), 3.87 (s, 6H), 3.76 (d, J
was stirred at 60 ꢁC for 36 hours. Excess salts were removed by ¼ 15.3 Hz, 4H), 3.68 (s, 6H), 3.46 (s, 12H), 3.30 (d, J ¼ 9.1 Hz,
centrifugation (4000 rpm for 10 min) and ltration; the ltrate 2H), 2.16 (d, J ¼ 11.9 Hz, 2H), 1.82 (d, J ¼ 8.1 Hz, 2H), 1.44–1.34
was concentrated in vacuo. The crude product was puried by (m, 2H), 1.12–1.03 (m, 2H). 13C NMR (101 MHz, CDCl3) d 167.0,
column chromatography (CombiFlash Rf automated column 165.4, 161.8, 159.8, 157.5, 149.1, 113.6, 109.6, 102.1, 90.1, 61.2,
system; 24 g HP silica; A: DCM, B: methanol, 100% A to 30% B 56.1, 55.4, 55.2, 53.5, 52.5, 52.2, 44.6, 26.0, 25.0. MS (ES+) m/z ¼
gradient) to afford the product as a brown oil (0.135 g, 80%). 1H 833.7 [M + H]+.
NMR signals were broad and could not be integrated. 13C NMR
(101 MHz, CDCl3) d 166.9, 165.2, 163.5, 148.8, 139.6, 129.1,
General procedure for methyl ester deprotection
128.0, 126.9, 111.8, 109.5, 59.3, 55.4, 53.5, 53.6, 46.4, 25.5, 24.5.
MS (ES+) m/z ¼ 653.6 [M + H]+.
To a stirred solution of methyl ester-protected ligand (13–17, 19)
Me2CHXdedpaOMe-N,N0-BnOMe (17). To a stirred solution of (approx. 60 mg) in THF/water (3 : 1, 4 mL), LiOH (12 mg, approx.
Me2CHXdedpaOMe (12) (0.39 g, 0.80 mmol) in CH3CN (8 mL) 5 equiv.) was added. The reaction mixture was stirred at
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ambient temperature for 1.5 h or until reaction progress was
complete (conrmed by absence of starting material peak in
MS, and disappearance of starting material spot on TLC (Rf
General procedure for Ga(III) complexation of pro-ligands (1–6)
A solution of pro-ligand (1–6) (0.02 mmol) in water/methanol
(1 : 1, 2 mL) was adjusted to pH 2–3 using 0.1 M HCl (aq). A
solution of Ga(ClO4)3$6H2O (0.03 mmol, 1.5 equiv.) in water
(500 mL) was added to this solution, and the pH was then
adjusted to 4–4.5 with 0.1 M NaOH (aq). The reaction mixture
was stirred at room temperature overnight, over which time
a white precipitate formed. The solid was subsequently isolated
by centrifugation (4000 rpm, 10 min) and dried further under
vacuum to yield the Ga-complex as a white solid (55–89% yield).
[Ga(dedpa-N,N0-ee)][ClO4], [Ga(1)][ClO4]. 1H NMR (400 MHz,
DMSO) d 8.66 (t, J ¼ 7.8 Hz, 2H), 8.35 (d, J ¼ 7.6 Hz, 2H), 8.19 (d,
J ¼ 7.9 Hz, 2H), 4.71 (d, J ¼ 17.6 Hz, 2H), 4.57 (d, J ¼ 17.5 Hz,
2H), 3.65 (t, J ¼ 4.8 Hz, 4H), 3.43 (q, J ¼ 14.1, 7.1 Hz, 5H), 3.37 (d,
J ¼ 11.1 Hz, 2H), 2.93 (d, J ¼ 11.1 Hz, 2H), 2.83 (t, J ¼ 4.6 Hz, 4H),
1.11 (t, J ¼ 7.0 Hz, 6H). 13C NMR (101 MHz, DMSO) d 162.2,
150.9, 146.4, 143.6, 128.2, 123.3, 65.8, 64.5, 56.3, 50.9, 48.5, 15.0.
HR ESI-MS m/z for C24H3269GaN4O6 (M+) calcd (found): 541.1584
(541.1578).
(product)
<
Rf (starting materials)) in 10% MeOH/
dichloromethane). The reaction mixture was concentrated in
vacuo and used without further purication, or puried by semi-
preparative RP-HPLC (A: CH3CN, B: 0.1% triuoroacetic acid
(TFA) in water, 5–100% A linear gradient, 25 min, 10 mL minꢀ1).
Product fractions were lyophilized to yield the ligand as a white
solid (80–99% yield).
H2dedpa-N,N0-ee (1). 1H NMR (300 MHz, MeOD) d 7.76 (d, J ¼
7.5 Hz, 2H), 7.68 (t, J ¼ 7.6 Hz, 2H), 7.20 (d, J ¼ 7.4 Hz, 2H), 3.71
(s, 4H), 3.24–3.16 (m, 4H), 3.18–3.06 (m, 4H), 2.29–2.24 (m, 4H),
2.23 (s, 4H), 0.86 (t, J ¼ 7.0 Hz, 6H). 13C NMR (75 MHz, MeOD)
d 172.2, 159.5, 154.5, 139.6, 125.5, 123.0, 68.1, 67.3, 61.3, 53.9,
51.6, 15.5. MS (ES+) m/z ¼ 475.3 [M + H]+, 481.3 [M + Li]+, 497.3
[M + Na]+.
1
H2CHXdedpa-N,N0-ee (2). H NMR (400 MHz, MeOD) d 8.10
(d, J ¼ 7.4 Hz, 2H), 8.01 (t, J ¼ 7.5 Hz, 2H), 7.80 (d, J ¼ 7.2 Hz,
2H), 4.48 (d, J ¼ 14.0 Hz, 2H), 4.06 (d, J ¼ 14.1 Hz, 2H), 3.79–3.66
(m, 4H), 3.56–3.48 (m, 2H), 3.41–3.33 (m, 2H), 3.27–3.16 (m,
4H), 3.16–3.05 (m, 2H), 2.38 (d, J ¼ 10.8 Hz, 2H), 1.99 (d, J ¼
6.4 Hz, 2H), 1.94–1.76 (m, 2H), 1.70–1.56 (m, 2H), 1.51–1.32 (m,
4H), 1.12 (s, 4H), 0.96 (t, J ¼ 6.5 Hz, 6H). MS (ES+) m/z ¼ 529.5 [M
+ H]+, 535.5 [M + Li]+.
[Ga(CHXdedpa-N,N0-ee)][ClO4], [Ga(2)][ClO4]. 1H NMR (400
MHz, DMSO) d 8.82 (t, J ¼ 7.8 Hz, 2H), 8.50 (d, J ¼ 7.6 Hz, 2H),
8.35 (d, J ¼ 7.9 Hz, 2H), 5.14 (d, J ¼ 18.2 Hz, 2H), 4.60 (d, J ¼
18.2 Hz, 2H), 3.64–3.37 (m, 8H), 3.19 (d, J ¼ 8.2 Hz, 2H), 3.09–
2.98 (m, 4H), 2.37 (d, J ¼ 11.3 Hz, 2H), 1.89 (d, J ¼ 7.0 Hz, 2H),
1.62–1.48 (m, 2H), 1.45–1.31 (m, 2H), 1.21 (t, J ¼ 7.0 Hz, 6H). 13
C
H2CHXdedpaOMe-N,N0-ee (3). 1H NMR (400 MHz, D2O, 55 ꢁC)
d 7.67 (s, 2H), 7.25 (s, 2H), 4.46 (d, J ¼ 13.0 Hz, 2H), 4.26 (d, J ¼
12.0 Hz, 2H), 4.16 (s, 6H), 4.09–3.97 (m, 4H), 3.89–3.79 (m, 4H),
3.76 (d, J ¼ 7.8 Hz, 2H), 3.49–3.34 (m, 4H), 2.53 (d, J ¼ 11.5 Hz,
2H), 2.21 (d, J ¼ 8.2 Hz, 2H), 1.88–1.74 (m, 2H), 1.66 (t, J ¼
9.9 Hz, 2H), 1.44 (t, J ¼ 7.0 Hz, 6H). 13C NMR (101 MHz, D2O, 55
ꢁC) d 172.1, 167.8, 156.6, 155.9, 118.4, 115.5, 111.9, 109.3, 67.0,
62.4, 56.1, 24.6, 24.4, 14.5. HR-ESI-MS m/z for C30H45N4O8 (M +
H)+ calcd (found): 589.3237 (589.3237).
NMR (101 MHz, DMSO) d 162.0, 151.9, 147.0, 144.0, 126.8,
123.6, 66.1, 65.9, 63.8, 57.6, 51.3, 48.8, 27.338, 23.9, 14.9. HR-
ESI-MS m/z for C28H3869GaN4O6 (M)+ calcd (found): 595.2047
(595.2048) (0.2 ppm).
[Ga(CHXdedpaOMe-N,N0-ee)][ClO4], [Ga(3)][ClO4]. 1H NMR
(300 MHz, DMSO) d 7.76 (s, 2H), 7.75–7.70 (m, 2H), 4.87 (d, J ¼
17.7 Hz, 2H), 4.27 (d, J ¼ 17.8 Hz, 1H), 4.11 (s, 6H), 3.39–3.16 (m,
8H), 2.99 (d, J ¼ 3.9 Hz, 2H), 2.88–2.75 (m, 2H), 2.16 (d, J ¼
9.7 Hz, 2H), 1.77–1.64 (m, 2H), 1.44–1.29 (m, 2H), 1.26–1.15 (m,
2H), 1.07 (t, J ¼ 6.9 Hz, 6H). 13C NMR (resonances obtained
from HSQC) (75 MHz, DMSO) d 111.5, 110.1, 66.3, 63.9, 58.3,
51.5, 51.1, 49.0, 27.7, 24.2, 15.3. HR-ESI-MS m/z for
H2CHXdedpaOMe-N,N0-Bn (4). 1H NMR (400 MHz, D2O, 55 ꢁC)
d 7.72 (dd, J ¼ 13.9, 6.2 Hz, 8H), 7.61–7.53 (m, 6H), 4.20 (s, 6H),
4.09 (d, J ¼ 15.4 Hz, 2H), 4.03–3.92 (m, 6H), 3.20 (d, J ¼ 6.7 Hz,
2H), 2.60 (d, J ¼ 11.0 Hz, 2H), 2.16 (d, J ¼ 6.9 Hz, 2H), 1.62–1.50
C
ppm).
30H4269GaN4O8 (M)+ calcd (found): 655.2258 (655.2253) (ꢀ0.8
13
ꢁ
(m, 4H). C NMR (101 MHz, D2O, 55 C) 129.8, 128.5, 127.5,
110.7, 108.2, 100.1, 60.0, 55.9, 54.6, 54.3, 25.9, 23.9. HR-ESI-MS
m/z for C36H41N4O6 (M + H)+ calcd (found): 625.3026 (625.3020).
H2CHXdedpaOMe-N,N0-BnOMe (5). 1H NMR (400 MHz, D2O, 75
ꢁC) d 8.22 (s, 2H), 7.63 (t, J ¼ 7.8 Hz, 2H), 7.54 (s, 2H), 7.47 (d, J ¼
7.3 Hz, 2H), 7.37 (s, 2H), 7.31 (d, J ¼ 8.3 Hz, 2H), 4.60 (d, J ¼
16.3 Hz, 2H), 4.55 (s, 6H), 4.38 (d, J ¼ 18.2 Hz, 2H), 4.33 (s, 4H),
4.13 (s, 6H), 3.84 (d, J ¼ 8.3 Hz, 2H), 2.86 (d, J ¼ 12.0 Hz, 2H),
2.47 (d, J ¼ 8.8 Hz, 2H), 2.10–1.97 (m, 2H), 1.93–1.80 (m, 2H).
13C NMR (101 MHz, D2O, 75 ꢁC) d 160.16, 153.42, 149.07, 131.06,
123.55, 116.97, 114.87, 113.66, 111.83, 100.12, 60.57, 57.75,
56.08, 55.36, 51.87, 24.52, 23.98. HR-ESI-MS m/z for C38H45N4O8
(M + H)+ calcd (found): 685.3237 (685.3231).
[Ga(CHXdedpaOMe-N,N0-Bn)][ClO4], [Ga(4)][ClO4]. 1H NMR
(300 MHz, DMSO) d 7.87–7.83 (m, 2H), 7.79 (d, J ¼ 1.6 Hz, 2H),
7.65 (d, J ¼ 6.8 Hz, 4H), 7.45–7.32 (m, 6H), 4.71 (br s, 4H), 4.17
(d, J ¼ 13.7 Hz, 2H), 4.14 (s, 6H), 3.46 (d, J ¼ 13.7 Hz, 2H), 3.25
(2, J ¼ 6.5 Hz, 4H), 1.32–1.21 (m, 2H), 1.20–1.06 (m, 2H), 1.03–
0.90 (m, 2H). 13C NMR (resonances obtained from HSQC) (75
MHz, DMSO) d 132.1, 128.7, 112.1, 110.2, 62.5, 62.1, 58.2, 49.3,
40.1. HR-ESI-MS m/z for C36H3869GaN4O6 (M)+ calcd (found):
691.2061 (691.2074) (2.0 ppm).
[Ga(CHXdedpaOMe-N,N0-BnOMe)][ClO4], [Ga(5)][ClO4]. 1H
NMR (600 MHz, DMSO) d 7.83 (d, J ¼ 6.0 Hz, 4H), 7.29 (t, J ¼
7.9 Hz, 2H), 7.25 (s, 2H), 7.19 (d, J ¼ 7.4 Hz, 2H), 6.99 (dd, J ¼
8.4, 1.7 Hz, 2H), 4.71 (d, J ¼ 17.8 Hz, 2H), 4.65 (d, J ¼ 17.8 Hz,
2H), 4.15 (s, 6H), 4.12 (d, 2H), 3.83 (d, 2H), 3.74 (s, 6H), 3.23–
3.17 (m, 2H), 1.29–1.20 (m, 4H), 1.16–1.07 (m, 4H). HR-ESI-MS
H2CHXdedpaOMe-N,N0-Bn3OMe (6). 1H NMR (400 MHz,
MeOD) d 7.65–7.43 (m, 4H), 5.88 (s, 4H), 3.91 (s, 6H), 3.68 (d, J ¼
26.7 Hz, 2H), 3.63 (s, 6H), 3.55 (d, 2H), 3.48 (s, 12H), 3.32 (d, J ¼
14.2 Hz, 4H), 2.55 (br s, 2H), 2.24–1.98 (m, 2H), 1.73–1.54 (m,
2H), 1.09–0.90 (m, 4H). HR-ESI-MS m/z for C42H52N4O12 (M +
H)+ calcd (found): 805.3660 (805.3658) (ꢀ0.2 ppm).
m/z for
(751.2266) (1.1 ppm).
C
38H4269GaN4O8 (M)+ calcd (found): 751.2258
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[Ga(CHXdedpaOMe-N,N0-Bn3OMe)][ClO4], [Ga(6)][ClO4]. 1H Human apo-transferrin stability studies
NMR (600 MHz, DMSO) d 7.82 (s, 2H), 7.80 (s, 2H), 6.19 (s, 4H),
The in vitro kinetic inertness assays of 67Ga-complexes against
human apo-transferrin were performed according to a previ-
ously published method.15,17,18 The radiolabelled complexes
[67Ga(2, 3, or 5)]+ were prepared with the radiolabeling protocol
as described above. In duplicate for each 67Ga-complex above,
solutions were prepared in vials with 500 mL of freshly prepared
1 mg mLꢀ1 apo-transferrin in NaHCO3 (10 mM, pH 7) solution,
300 mL of 67Ga-complex, and 200 mL of phosphate buffered
saline (PBS, pH 7.4), and incubated at 37 ꢁC in a water bath. At
time points 15 min, 1 and 2 hours, 300 mL of the apo-transferrin
competition mixture was removed from each vial, diluted to
a total volume of 2.5 mL with PBS, and then counted in a Cap-
intec CRC 15R well counter to obtain a value for the total activity
to be loaded on the PD-10 column (F). The 2.5 mL of diluted
apo-transferrin mixture was loaded onto a PD-10 column that
had previously been conditioned via elution with 20 mL of PBS,
and the empty vial was counted in a well counter to determine
the residual activity le in the vial (R). The 2.5 mL of loading
volume was allowed to elute into a waste container, and then
the PD-10 column was eluted with 3.5 mL of PBS and collected
into a separate vial. The eluent which contained 67Ga bound/
associated with apo-transferrin (size exclusion for MW < 5000
Da) was counted in a well counter (E) and then compared to the
total amount of activity that was loaded on the PD-10 column to
obtain the percentage of 67Ga that was bound to apo-transferrin
and therefore no longer chelate-bound using the equation:
4.43 (dd, J ¼ 17.8 Hz, 4H), 4.14 (s, 6H), 4.09 (d, J ¼ 14.0 Hz, 2H),
3.76 (s, 12H), 3.69 (s, 6H), 3.29 (d, J ¼ 14.5 Hz, 2H), 2.90 (d, J ¼
6.2 Hz, 2H), 1.49–1.38 (m, 4H), 1.16–1.05 (m, 2H), 0.88–0.78 (m,
2H).13C NMR (151 MHz, DMSO) d 173.4, 162.3, 162.0, 159.7,
153.1, 146.5, 111.8, 109.6, 101.4, 90.8, 65.4, 58.0, 56.0, 55.5, 50.9,
49.7, 29.5, 24.5. HR ESI-MS m/z for C42H5069GaN4O12 (M+) calcd
(found): 871.2681 (871.2684) (0.3 ppm).
67/68Ga radiolabeling studies
The 67Ga radiolabeling protocol of all pro-ligands followed
those previously described.16,17 Briey, for initial labeling
studies and human apo-transferrin challenge assays, all chela-
tors were made up as stock solutions (1 mg mLꢀ1, ꢄ10ꢀ3 M) in
deionized water. A 100 mL aliquot of each chelator stock solu-
tion was transferred to screw-cap mass spectrometry vials and
diluted with pH 4 NaOAc (10 mM) buffer such that the nal
volume was 1 mL aer the addition of 67GaCl3, to a nal
chelator concentration of ꢄ10ꢀ4 M for each sample. An aliquot
of 67GaCl3 (ꢄ1 mCi for labeling studies and log P determina-
tion, and ꢄ3–6 mCi for apo-transferrin competitions) was
added to the vials containing the chelator and allowed to
radiolabel at ambient temperature for 10 min, then analyzed by
RP-HPLC to conrm radiolabeling and calculate yields. Areas
under the peaks observed in the HPLC radiotrace were inte-
grated to determine radiolabeling yields. Elution conditions
used for RP-HPLC analysis were gradient: A: 10 mM NaOAc
buffer, pH 4.5, B: CH3CN; 0 to 100% B linear gradient 20 min.
For 68Ga radiolabeling studies, selected pro-ligands were
prepared as above (1 mg mLꢀ1, ꢄ10ꢀ3 M) in deionized water. A
100 mL aliquot of each chelator stock solution was transferred to
screw-cap mass spectrometry vials and diluted with pH 5.5
NaOAc (100 mM) buffer such that the nal volume was 1 mL
aer the addition of 68GaCl3 (50–100 mL), to a nal chelator
concentration of 10ꢀ4 M and reaction pH of 4.5. Reaction vials
were le to sit at ambient temperature for 10 minutes, and
analysed by RP-HPLC as above for 67Ga reactions. Elution
conditions used for RP-HPLC analysis of 68Ga reactions were
gradient: A: 0.1% TFA (triuoroacetic acid), B: CH3CN; 0 to
100% B linear gradient 20 min.
ꢀ
ꢁ
E
1 ꢀ
ꢃ 100:
ðF ꢀ RÞ
Conclusions
The synthesis and characterization of six new lipophilic deriv-
atives of H2dedpa or H2CHXdedpa for the intended purpose of
myocardial perfusion imaging were described. All six derivatives
were screened for their ability to bind Ga(III) and retained hex-
adentate N4O2 binding. Initial radiolabeling experiments with
67Ga resulted in abnormal radiolabeling HPLC chromatograms
in which three 67Ga-labelled products were formed for the N,N0-
benzyl functionalized ligand derivatives (4–6), while ligands (1–
3), with N,N0-ethyl ether moieties, exhibited single peaks in the
radio-chromatogram as expected. In an attempt to further
log P determination
The log P of 67Ga-complexes were measured experimentally identify the additional by-product peaks in the radiolabeling
following procedures outlined previously.17 Briey, 67Ga- reactions of [67/68Ga(4–6)]+ as either ligand impurities or ligand
labelled complex (30 mCi/6 mL) was added to 1.5 mL centrifuge radiolysis products, reaction mixture [67Ga(5)]+ was analysed
tubes containing phosphate buffered saline (PBS) (494 mL) and over the course of one hour, and pro-ligand 6 was radiolabelled
1-octanol (500 mL), mixed vigorously for 1 minute, and subse- with 68Ga as well as 67Ga. The relative peak intensities of the
quently centrifuged to separate phases (3000 rpm, 5 min). Equal three radiolabelled products remained constant despite the
aliquots (20–100 mL) of the organic and aqueous phases were changing reaction parameters, suggesting ligand impurities
diluted in a standard volume (20 mL) of water for measurement present in even low concentrations are able to complex Ga(III)
in an N-type Co-axial HPGe gamma spectrometer from Canberra more efficiently than the desired pro-ligand. Even though the
tted with a 0.5 mm beryllium window and calibrated (energy ligands were deemed sufficiently pure post purication via
and efficiency) with a 20 mL 152Eu source. The samples were HPLC and NMR characterization, the relative steric-bulk
counted for 10 minutes, with a dead time of less than 5%. imposed by benzyl groups next to the cyclohexane backbone
Measurements were carried out in triplicate.
may be conceivably inducing ligand decomposition overtime.
103772 | RSC Adv., 2016, 6, 103763–103773
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Therefore, ligands 4–6 are not suitable candidates as radio-
pharmaceuticals, as a major requirement of such pharmaceu-
ticals is that they be chemically stable and have a suitable shelf-
life.
3 R. S. Gibson, D. D. Watson, G. B. Craddock, R. S. Crampton,
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4 S. Liu, Dalton Trans., 2007, 1183–1193.
Partition coefficients of CHXdedpa2ꢀ N,N-ethyl ether func-
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of ꢀ1.5 to ꢀ1.8, and are not in the predicted suitable log P range
for myocardial perfusion imaging agents. The 67Ga-dedpa
complex ([Ga(1)]+, [Ga(dedpa-ee)]+) exhibited a log P nearly one
full log unit lower than the analogous 67Ga-CHXdedpa complex
([Ga(2)]+, [Ga(CHXdedpa-ee)]+), ꢀ2.7 and ꢀ1.8, respectively. This
nding suggests that H2CHXdedpa presents a potentially useful
framework to design lipophilic ligands in future studies. More-
over, selected 67Ga-CHXdedpa complexes were sufficiently stable
(>80%) in a 2 h apo-transferrin challenge assay.
5 L. Maria, C. Fernandes, R. Garcia, L. Gano, A. Paulo,
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¨ ¨
10 M. Tarkia, A. Saraste, T. Saanijoki, V. Oikonen, T. Vahasilta,
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attachments was an unfortunate choice of appendage which
led to non-trivial radiolabeling and potentially unstable ligands,
M. Strandberg, C. Stark, T. Tolvanen, M. Teras, T. Savunen,
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Acknowledgements
We acknowledge Nordion (Canada) and the Natural Sciences
and Engineering Research Council (NSERC) of Canada for grant
support (CR&D, Discovery), NSERC for a CGS-M/CGS-D fellow-
ships (C. F. R.), and the University of British Columbia for 4YF
fellowships (C. F. R.). C. O. acknowledges the Canada Council
for the Arts for a Killam Research Fellowship (2011–2013). The
authors also wish to thank Nordion for supplying 67GaCl3 and
17 C. F. Ramogida, J. Pan, C. L. Ferreira, B. O. Patrick,
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RSC Adv., 2016, 6, 103763–103773 | 103773