Synthesis, gallium-68 radiolabelling and biological evaluation of a series of triarylphosphonium-functionalized DO3A chelators

Article abstract of DOI:10.1039/C8DT02966K

Radiolabelled lipophilic cations that accumulate in mitochondria according to the magnitude of the mitochondrial membrane potential can be used to report non-invasively on mitochondrial dysfunction in cardiovascular disease, cardiotoxicity, and cancer. While several such cations are already commercially available for SPECT imaging, PET offers greater promise in terms of sensitivity, resolution, and capacity for dynamic imaging and pharmacokinetic modelling. We have therefore synthesised a series of three triarylphosphonium-functionalised DO3A chelators for positron emitter gallium-68, with differing alkyl-functionalisation motifs to provide opportunities for tunable lipophilicity as a means of optimising their pharmacokinetics. To assess their capacity to report on mitochondrial membrane potential, we assessed their pharmacokinetic profiles in isolated tumour cells and isolated perfused rat hearts before and after mitochondrial depolarisation with the ionophore CCCP. All three compounds radiolabelled with over 97% RCY and exhibited log?D values of between ?3.12 and ?1.81. In vitro assessment of the uptake of the radiotracers in cultured tumour cells showed a three-fold increase in uptake compared to unchelated [⁶⁸Ga]Ga(iii). However, each complex exhibited less than 1% retention in healthy hearts, which was not significantly diminished by mitochondrial depolarisation with CCCP. This preliminary work suggests that while this approach is promising, the lipophilicity of this class of tracers must be increased in order for them to be useful as cardiac or cancer imaging agents.

Full text of DOI:10.1039/C8DT02966K

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PAPER
Synthesis, gallium-68 radiolabelling and biological
evaluation of a series of triarylphosphonium-
functionalized DO3A chelators†
Cite this: Dalton Trans., 2018, 47,
15448
a,b
a,b
b
b
Adam J. Smith,
Peter J. Gawne,
Michelle T. Ma,
Philip J. Blower,
b
a
Richard Southworth * and Nicholas J. Long
*
Radiolabelled lipophilic cations that accumulate in mitochondria according to the magnitude of the mito-
chondrial membrane potential can be used to report non-invasively on mitochondrial dysfunction in
cardiovascular disease, cardiotoxicity, and cancer. While several such cations are already commercially
available for SPECT imaging, PET offers greater promise in terms of sensitivity, resolution, and capacity for
dynamic imaging and pharmacokinetic modelling. We have therefore synthesised a series of three triaryl-
phosphonium-functionalised DO3A chelators for positron emitter gallium-68, with differing alkyl-func-
tionalisation motifs to provide opportunities for tunable lipophilicity as a means of optimising their phar-
macokinetics. To assess their capacity to report on mitochondrial membrane potential, we assessed their
pharmacokinetic profiles in isolated tumour cells and isolated perfused rat hearts before and after
mitochondrial depolarisation with the ionophore CCCP. All three compounds radiolabelled with over
9
7% RCY and exhibited log D values of between −3.12 and −1.81. In vitro assessment of the uptake of the
radiotracers in cultured tumour cells showed a three-fold increase in uptake compared to unchelated
6
8
Received 20th July 2018,
[ Ga]Ga(III). However, each complex exhibited less than 1% retention in healthy hearts, which was not
Accepted 3rd October 2018
significantly diminished by mitochondrial depolarisation with CCCP. This preliminary work suggests that
while this approach is promising, the lipophilicity of this class of tracers must be increased in order for
them to be useful as cardiac or cancer imaging agents.
DOI: 10.1039/c8dt02966k
rsc.li/dalton
1
,2
increases 10-fold for every 61.5 mV increase in ΔΨm. This
means that the degree of uptake of lipophilic cations can be
Introduction
Lipophilic cations are widely used to map myocardial per- related to mitochondrial function or dysfunction; for example,
fusion by means of SPECT, and to a lesser extent, PET compared to healthy mitochondria, ΔΨm is reduced to below
imaging. They can accumulate in the mitochondrial matrix in 100 mV in myocardial tissue recovering from reperfusion
a manner dependent on mitochondrial membrane potential injury, and at least 60 mV higher in tumour cells, and the
5
,6
(
ΔΨ ), which in normally functioning mitochondria is uptake of lipophilic cations varies accordingly.
m
1
–4
between 150–180 mV.
In lipophilic cations that have been
This basic concept has long been used experimentally to
successfully used as probes of ΔΨ , the charge is delocalised report on ΔΨm through the use of fluorescent lipophilic
m
over a large molecular surface area and as such, the tracers cations such as tetramethylrhodamine (TMRE), BODIPY and
2
7–9
can pass easily through the lipid bilayer membrane.
Application of the Nernst equation to monocations predicts culture and isolated tissue preparations, however the poor
00- to 500-fold accumulation compared to the extracellular depth penetration and lack of whole body scanning inherent
Rhodamine-123.
These tracers show great utility in cell
1
space, and at 37 °C, mitochondrial uptake of lipophilic cations to fluorescent tracers limits their overall utility. On the other
hand, nuclear medicine imaging methodologies such as
SPECT and PET provide unparalleled depth penetration, allow-
ing whole body imaging; clinically, SPECT-based lipophilic
a
Department of Chemistry, Imperial College London, Molecular Sciences Research Hub,
White City Campus, 80 Wood Lane, W12 0BZ, UK. E-mail: n.long@imperial.ac.uk
9
9m
99m
cations such as [ Tc]Tc-sestaMIBI (Fig. 1) and [ Tc]Tc-
b
School of Biomedical Engineering and Imaging Sciences, King’s College London,
10–13
tetrofosmin (t
1
2
= 6 h) are used.
A clinical PET “equivalent”
4
th Floor Lambeth Wing, St. Thomas’ Hospital, London, SE1 7EH, UK.
E-mail: richard.southworth@kcl.ac.uk
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8dt02966k
99m
to the SPECT radiopharmaceuticals [ Tc]Tc-sestaMIBI and
9
9m
[
Tc]Tc-tetrofosmin would enable higher resolution
†
imaging, and potential detection of anatomically smaller
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2
8–30
agents.
Unlike cyclotron-produced copper-64, gallium-68
is generator-produced, and so has the potential to be more
3
1–34
widely used as generators become clinically widespread.
There has been some preliminary work on synthesising
gallium-labelled lipophilic cations for mitochondrial imaging
e.g. Kardashinsky et al. have synthesised DO3A-xy-TAP chela-
tors for gallium-67, in which the TAP groups exhibit methyl
3
5–37
and pyridyl functionalisation.
However, no mitochondrial
uptake studies have been performed on these chelators, and
their utility as gallium-68 based imaging agents has yet to be
explored.
In this study, we present a series of DO3A chelators for
gallium-68 complexation, functionalised with triphenylpho-
sphonium, tritolylphosphonium (TTP) and trixylylphospho-
nium (TXP) (Fig. 2). These chelators are designed to combine
the rationale behind the DO3A-xy-TPP chelators previously
used for copper-64 and gallium-67 chelation, with tunable
lipophilicity afforded by alkyl-functionalised triarylphospho-
nium cations in order to form the novel chelators DO3A-xy-
2
7,29,37
TTP and DO3A-xy-TXP.
After initial synthesis and charac-
terisation of the ligands, their radiolabelling characteristics
and log D values were analysed to give an indication of their
potential use as mitochondrial imaging agents. The tracers
9
9m
18
Fig. 1
[
Tc]Tc-sestaMIBI and [ F]MitoPhos_07, two examples of
10,27
lipophilic and cationic radiotracers.
tissue defects with aberrant ΔΨ . In addition, PET radiotracers
m
allow for dynamic imaging, so that the kinetics of influx and
efflux can be easily followed. It should also be noted that there
exist examples of multimodal optical/PET imaging agents,
which consist of a fluorophore functionalised with a PET
1
4–16
radioisotope.
These compounds allow for the combination
of the high resolution of optical imaging with the depth pene-
tration of PET imaging.
PET radioisotopes such as carbon-11 and fluorine-18 (t =
1
2
2
0 and 109.7 min respectively) have previously been incorpor-
ated into lipophilic triphenylphosphonium (TPP) cations. TPP
is widely known to target mitochondria since the cationic
charge nominally residing on the phosphorus atom can be
3
,4,17,18
delocalised around the aromatic rings.
The first PET
tracer to utilise TPP was designed by Fukuda et al. and con-
1
9
sisted of TPP methylated with carbon-11 (methylTPP). Since
then, there have been many different types of TPP-functiona-
lised PET tracers for mitochondrial imaging, most commonly
using fluorine-18 since its longer half-life facilitates clinical
2
0–25
imaging.
Recently, our group has synthesised the
MitoPhos series of tracers, which are fluorine-18 radiolabelled
alkyl-functionalised triarylphosphonium (TAP) cations, in order
to provide radiotracers with tunable lipophilicity. One example
1
8
26,27
of this series is [ F]MitoPhos_07, shown in Fig. 1.
Metallic PET radioisotopes, such as copper-64 and gallium-
8 (t = 12.7 h and 68 min respectively) have the advantage that
6
1
2
once the chelator has been synthesised, the radiometal can be
added via a simple final synthetic step. Chelators such as
DO3A-xy-TPP, which consist of a DO3A chelating moiety,
a xylyl linking group (xy) and a TPP cation, have been shown
Fig. 2 The three gallium-68 chelate complexes synthesised in this
study: [ Ga]Ga-DO3A-xy-TPP, [ Ga]Ga-DO3A-xy-TTP and [ Ga]Ga-
68
68
68
to chelate copper-64 to form effective tumour imaging DO3A-xy-TXP.
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4
2
were biologically evaluated in cultured tumour cells and using mide to form TAP-functionalised DO3A derivatives (3). Using
the ex vivo Langendorff isolated perfused heart model with a sodium carbonate as an inorganic base allowed for the purifi-
unique triple gamma detector system, which has been used cation by recrystallisation of the tris-tert-butyl protected inter-
previously
pharmacokinetics.
in
our
group
to
assess
radiotracer mediate, as well as its characterisation by NMR. The final step
was deprotection of the tert-butyl protected carboxylates (3)
using trifluoroacetic acid, followed by purification by semipre-
parative reverse-phase HPLC.
3
8–41
Results
Synthesis of the DO3A-xy-TAP chelators
6
8
Radiolabelling studies of DO3A-xy-TAP with [ Ga]GaCl eluate
3
All three DO3A-xy-TAP compounds were radiolabelled and ana-
lysed using the same protocol. The ligand was dissolved in
aqueous ammonium acetate solution in order to partially neu-
tralise the acidic generator eluate containing [ Ga]Ga(III).
Next, the [ Ga]Ga(III) eluate was added, and the resulting
mixture was heated at 100 °C for 30 minutes. As a quality
control (QC) method, instant thin-layer chromatography (iTLC)
was employed, using a mobile phase of 0.1 M EDTA, in which
unchelated [ Ga]Ga(III) migrated to the solvent front, and left
the cationic complex, which resisted transchelation to EDTA,
on the baseline. iTLC analysis gave RCY values of 98, 100 and
7% for 100 μM concentrations of [ Ga]Ga-DO3A-xy-TPP, TTP
and TXP respectively. Due to the high RCY of the three radiola-
belling processes, no post-purification was undertaken.
Finally, the reaction mixture was analysed by radioHPLC to
determine whether a single complex had formed and to give
an initial indication of its lipophilicity. The radioHPLC traces
obtained for the three compounds are shown in normalised
form in Fig. 3. In each trace, a small peak can be seen at
approximately 2 minutes, which corresponds to unreacted/
DO3A-xy-TAP chelators were synthesised as outlined in
Scheme 1. The synthesis combined methodologies used by
Haslop et al., Wang et al. and Kardashinsky et al.
an alkyl-functionalised aryl bromide was converted into a
Grignard reagent before being reacted with phosphorus tri-
chloride to form a triarylphosphine (1). This strategy allowed
tuning of the final tracer lipophilicity by altering the alkyl
functionalisation, in accordance with the work performed by
2
7,28,37
Firstly,
68
68
68
2
7
Haslop et al. Whilst anaerobic and anhydrous conditions
were maintained as far as possible, some triarylphosphine was
3
1
1
found by P{ H}-NMR to be oxidised to its phosphine oxide.
The phosphine oxide impurity did not require additional puri-
fication, since it was inert during the subsequent nucleophilic
substitution step due to the lack of a phosphorus-centred lone
pair.
68
9
Secondly, the triarylphosphine was reacted with α,α′-
dibromo-m-xylene to form the phosphonium compound and
incorporate the xylyl linking group (2). Wang et al. performed
this reaction at 100 °C in toluene, forming a mixture of the
desired monosubstituted product as well as starting material
and disubstituted impurities. Instead, we performed the
reaction at the lowest temperature at which the dibromoxylene
fully dissolves in toluene, i.e. 60 °C. At this temperature, as 2
forms, it precipitates out of the non-polar toluene solvent, and
thus can be readily isolated by vacuum filtration.
68
non-chelated [ Ga]Ga(III) species, and a single major peak
2
8
corresponding to the complex, showing no evidence of isomer-
ism. The relative retention times of the three compounds
provide an insight into the lipophilicities of the three com-
pounds. As can be seen from Fig. 2, increased TAP alkylation
results in a longer retention time on a reverse-phase C-18
column, and indicates relatively greater lipophilicity. This
trend is also seen in analogous HPLC traces of the unchelated
The monocationic bromoxylene phosphonium derivative 2
was then reacted with tris-tert-butyl-protected DO3A hydrobro-
t
Scheme 1 Synthesis of DO3A-xy-TAP chelators. DO3A tris- Bu·HBr was
4
2
synthesised according to previously reported procedures. Reaction
conditions: (i) Mg then PCl , dry THF, anaerobic conditions; (ii) α,α’-
3
dibromo-p-xylene, toluene, 60 °C; (iii) DO3A tris- Bu·HBr, Na CO ,
MeCN, reflux; (iv) TFA, semi-prep HPLC. The choice of the aryl bromide
determines which DO3A-xy-TAP variant is synthesised: Ar = phenyl,
DO3A-xy-TPP; Ar = tolyl, DO3A-xy-TTP; Ar = m-xylyl, DO3A-xy-TXP.
6
8
3
Fig. 3 RadioHPLC traces of the three labelled [ Ga]Ga-DO3A-xy-TAP
compounds (eluent gradient: 100% A for 5 min; 0–80% B in A for 20 min;
100% A for 5 min; flow rate 1 mL min− ). All traces have been normalised
such that the maximum activity measured for each compound is equal
to 1.
t
2
1
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Table 1 Retention times (in minutes) of the unchelated DO3A-xy-TAP
ligands, along with their gallium-68-radiolabelled analogues as
measured by HPLC. HPLC conditions are identical to those described in
Fig. 3
6
8
Ar=
Unchelated ligand
[ Ga]Ga complex
TPP
TTP
TXP
14.3
17.2
18.5
14.8
17.6
19.9
ligands; the retention times of which are summarised in
Table 1. The unchelated ligands also show shorter HPLC reten-
tion times by HPLC than their gallium-68-labelled counter-
parts, which indicates an increase in lipophilicity upon chela-
tion, a result of the hydrophilicity of the carboxylate arms
decreasing upon binding to the metal centre.
6
8
Lipophilicity measurements of [ Ga]Ga-DO3A-xy-TAP
compounds
6
8
Fig. 5 Results of the in vitro tumour cell uptake experiment for [ Ga]
6
8
Ga-DO3A-xy-TXP and unchelated [ Ga]Ga(III) control. Measurements
To measure the lipophilicities of the radiotracers, their
octanol/PBS partition coefficients at pH 7.4 (log D) were deter-
mined using a conventional ‘shake-flask’ method. The log D
values, shown in Fig. 4, were determined to be −3.12 ± 0.17,
were taken in triplicate. Data are mean ± SD. * = P < 0.05.
68
Quantification of [ Ga]Ga-DO3A-xy-TXP myocardial uptake
6
8
−2.66 ± 0.09 and −1.81 ± 0.02 for [ Ga]Ga-DO3A-xy-TPP, TTP
using the Langendorff isolated perfused heart model
and TXP respectively (n = 6 for all compounds).
We have previously made use of the Langendorff isolated per-
fused heart model to provide an ex vivo assessment of myocar-
dial radiotracer uptake, with a unique triple radiodetector
6
8
In vitro quantification of [ Ga]Ga-DO3A-xy-TXP uptake in
tumour cells
system which allows for accurate determination of time-activity
6
8
Intracellular/extracellular concentration ratios for [ Ga]Ga-
38–41
curves.
The time-activity curves for each of the three radio-
6
8
DO3A-xy-TXP and unchelated [ Ga]Ga(III) in breast cancer cells
MDA-MB 231) in vitro were calculated, and are summarised in
detectors quantify radiotracer entering the heart (arterial
detector), radiotracer retention in the heart (heart detector)
and radiotracer exiting the heart (venous detector). The ex vivo
nature of the experiment means that the behaviour of the
tracer in the myocardium can be evaluated accurately and
reproducibly without the complications of the in vivo setting
such as aberrant metabolism or regional changes in perfusion,
and haemodynamic parameters can be easily monitored,
whilst maintaining constant perfusion to determine the
specific contribution of mitochondrial membrane potential to
tracer pharmacokinetics.
(
6
8
Fig. 5. [ Ga]Ga-DO3A-xy-TXP shows a ratio of 10.03 ± 0.45,
indicating that the tracers localise inside the tumour cells. In
6
8
comparison, [ Ga]Ga(III) shows a ratio of 3.43 ± 0.39, indicat-
ing that the chelator facilitates a three-fold increase in radio-
6
8
tracer uptake compared to unchelated [ Ga]Ga(III). A trypan
blue cell viability study showed over 90% of cells used in both
6
8
the [ Ga]Ga-DO3A-xy-TXP and control experiments survived.
To assess radiotracer uptake under conditions in which
ΔΨ
m
is reduced, the heart was infused with a 600 nM solution
of the ionophore carbonyl cyanide m-chlorophenyl hydrazone
4
3
(
CCCP). We have previously found that this concentration is
optimal to effect complete ΔΨ depolarisation, validated
m
99m
using the clinical SPECT radiopharmaceutical, [ Tc]Tc-
sestaMIBI (Fig. 1) to ensure validity of the model for this set
4
4
of experiments. As shown in Fig. 6A and summarised in
9
9m
Fig. 6B, [ Tc]Tc-sestaMIBI demonstrates 30 ± 0.03% (n = 4)
radiotracer retention after 15 minutes in hearts under normal
conditions, but 0 ± 0.04% (n = 4) retention after 15 minutes in
1
2
hearts under depolarising conditions.
6
8
[
Ga]Ga-DO3A-xy-TXP (1 MBq) was injected as a bolus into
6
8
the flow of Krebs buffer. Arterial influx, myocardial retention
and venous efflux of radiotracer were measured using each
Fig. 4 Log D values obtained of the three labelled [ Ga]Ga-DO3A-xy-
TAP compounds. Data are mean ± SD (n = 6).
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68
Fig. 6 (A and C) Representative example of data output of the triple-gamma detector system for [
Tc]Tc-sestaMIBI and [ Ga]Ga-DO3A-xy-TXP
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9m
68
respectively. (B and D) Retention values obtained using the two-injection protocol 15 minutes post-injection for [
DO3A-xy-TXP respectively. Control data were obtained from the injection before CCCP infusion, whilst CCCP data were obtained during 600 nM
Tc]Tc-sestaMIBI and [ Ga]Ga-
9
9m
68
CCCP infusion. Data for [
Tc]Tc-sestaMIBI are mean ± SD (n = 4). * = P < 0.05. Data for [ Ga]Ga-DO3A-xy-TXP are mean ± SD (n = 3).
respective γ-detector (Fig. 6A and C) for 15 min. Then, to approach, which allows a library of compounds to be syn-
assess radiotracer uptake under depolarising conditions, the thesised using identical reaction protocols. Use of a lower reac-
same heart was infused with CCCP. After 5 min of infusion tion temperature during formation of the phosphonium cation
6
8
with CCCP, the second bolus of [ Ga]Ga-DO3A-xy-TXP was allows for the facile isolation of the pure target compound,
injected, and the passage of radioactivity through the per- which is especially useful given the difficulties in purifying
fusion system was monitored using the three γ-detectors. As phosphonium cations using column chromatography.
6
8
shown by Fig. 6C, most of the injected [ Ga]Ga-DO3A-xy-TXP
Upon radiolabelling, the three gallium-68 radiolabelled
washed out of the heart within 3 minutes of bolus adminis- compounds synthesised all show high RCY values above 97%,
tration, irrespective of whether the mitochondria were despite the size mismatch between DO3A and Ga(III) cations.
depolarised. This stands in contrast to the control data for Whilst DO3A is not an ideal chelator for Ga(III) cations since
9
9m
mitochondrial depolarisation dependant uptake of [ Tc]Tc- radiolabelling requires heating and lengthy reaction times, it
sestaMIBI discussed previously. The average retained gallium- was chosen for the simplicity of its functionalisation chem-
6
8 activity between 14–15 minutes post-injection over 3 experi- istry. A useful part of this work is the method for the QC of
6
8
ments is reported in Fig. 6D. [ Ga]Ga-DO3A-xy-TXP showed gallium-68 labelled lipophilic cations. Basing this method on
% retention in both control and CCCP-infused hearts. Over 3 the facile chelation of Ga(III) by EDTA means that this method
experiments, there was no statistically significant difference should be easily applicable to future syntheses of all gallium-
1
6
8
between [ Ga]Ga-DO3A-xy-TXP retention in control and CCCP 68 labelled lipophilic cations.
infused hearts.
Functionalisation of the TAP moiety with alkyl groups has
been shown to dramatically affect the lipophilicity of the radio-
tracers with a 0.5 unit difference between the TPP and TTP-
functionalised compounds, and a 0.8 unit difference between
the TTP and TXP-functionalised compounds. It is interesting
Discussion
The series of DO3A-xy-TAP compounds was designed to be that even when conjugated to the bulky and hydrophilic DO3A
simple to synthesise and purify, whilst also being easy to chelator, such a small change as the addition of three methyl
modify in order to generate a series of compounds with groups to the TPP group has such a dramatic effect on the
tunable lipophilicity. Unlike other syntheses of TAP-functiona- log D values. The log D values of the compounds are low when
lised DO3A compounds, this work utilises a highly modular compared to other lipophilic cationic compounds such as
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9
9m
45
[
Tc]Tc-sestaMIBI, which has a log D value of 1.29.
0.1% TFA. Semi-preparative reverse phase HPLC was con-
Nevertheless, it was decided to assess the tracer ex vivo as ducted using an Agilent Eclipse XDB-C18 column (21.2 ×
there is precedent of cations with low log D values crossing 150 mm, 5 μm) with a 5 mL min− flow rate and UV spectro-
1
2
5,46
lipid bilayer membranes.
scopic detection at 214 nm. Analytical reverse phase HPLC was
6
8
Uptake experiments with [ Ga]Ga-DO3A-xy-TXP in tumour conducted using an Agilent Eclipse XDB-C18 column (4.6 ×
cells were undertaken in vitro, due to the high ΔΨ
often found in tumour cells.
DO3A-xy-TXP showed a three-fold increase in uptake in the [ Ga]GaCl from an Eckert and Ziegler gallium-68 generator
cancer cell line when compared to unchelated [ Ga]Ga(III), using a 0.1 M solution of hydrochloric acid.
indicating that the chelator facilitates uptake of gallium-68
values 150 mm, 5 μm) with a 1 mL min− flow rate and UV spectro-
1
m
68
1
,2,6,47–49
In this study, [ Ga]Ga- scopic detection at 250 nm. Gallium-68 was eluted as
6
8
3
6
8
Synthesis
into tumour cells.
Analysis using the perfused heart model showed no statisti- Synthesis of tris(3,5-dimethylphenyl)phosphane (1c) was
6
8
cally significant difference in [ Ga]Ga-DO3A-xy-TXP retention performed according to previously reported literature pro-
2
7
between healthy and CCCP-infused hearts. In addition, the cedures by Haslop et al. Synthesis of 1,4,7-tris(tert-butoxy-
cardiac uptake of our compounds was low when compared to carbonylmethyl)-1,4,7,10-tetraazacyclododecane hydrobromide
9
9m
the parallel study performed using [ Tc]Tc-sestaMIBI. This (DO3Atriester·HBr) was performed according to previously
6
8
42
is likely a result of the lower lipophilicity of [ Ga]Ga-DO3A-xy- reported literature procedures by Jagadish et al.
TXP hindering passage through the lipid bilayer membrane. Tri-p-tolylphosphane (1b). This compound was originally
Since the membrane potential in a normally functioning heart synthesised by Mann et al. The methodology used here was a
5
0
2
7
does not facilitate uptake of these tracers in cardiac cells, there modification of the procedure described by Haslop et al.
will then be no difference in uptake once the mitochondria A solution of 4-bromotoluene (2.89 mL, 23.5 mmol) in an-
have been depolarized by CCCP infusion. This preliminary hydrous THF (10 mL) was added dropwise whilst maintaining
work suggests that while this approach is promising from a reflux to magnesium turnings (0.57 g, 23.5 mmol) in a Schlenk
synthetic strategy perspective, the lipophilicity of this class of flask under N
tracers must be increased in order for them to be useful as before a solution of PCl
2
. The resulting suspension was stirred for 2 h
(0.69 mL, 7.8 mmol) in anhydrous
3
cardiac or cancer imaging agents.
THF (15 mL) was added dropwise whilst maintaining reflux,
and the resulting mixture was stirred for a further 2 h. The
reaction was quenched with water (10 mL), and the organic
phase was isolated. The organic solvent was removed under
Conclusions
reduced pressure to yield the title compound as a pale yellow
1
We have synthesised a series of TAP-functionalised DO3A solid (0.96 g, 40%). H-NMR (400 MHz, CDCl
3
) δ
).
H
1
(ppm): 7.24
C{ H}-NMR
3
1
ligands for gallium-68 chelation with a view to synthesising (6H, m), 7.17 (6H, m), 2.38 (9H, s, CH3
lipophilic cationic radiotracers. The synthetic methodology (100 MHz, CDCl ) δ
P{ H}-NMR (162 MHz, CDCl
basis of a strategy for synthesising libraries of tracers with (ES-TOF+): m/z calcd for C H P ([M + H] ) 305.1459, found
̲
3
c
(ppm): 138.6, 133.8, 129.4, 129.3, 21.4.
) δ (ppm): −7.9. HRMS
3
1
1
developed is effective and entirely modular, and can be the
3
P
+
2
1
22
tunable lipophilicities, by varying the chelator, linker and cation. 305.1445.
The results encourage the development of future gallium-68
General procedure 1: monosubstitution of α,α′-dibromo-p-
xylene with aryl-functionalized triarylphosphines (2)
labelled lipophilic cations with chelators more appropriate for
the efficient formation of lipophilic and positively charged
complexes of gallium-68.
The methodology used was a modification of the procedure
2
8
described by Wang et al. α,α′-Dibromo-p-xylene (1 eq.) was
added to toluene (5 mL) and heated to 60 °C until all the solid
had dissolved. To this, a solution of the triarylphosphine
Experimental procedures
Materials and instrumentation
(
1 eq.) in toluene (6 mL) was added dropwise, before the result-
ing solution was stirred at 60 °C under an N atmosphere over-
2
Commercially available reagent grade solvents and chemicals night. The white precipitate was isolated by filtration, before
were used without further purification. All gas mixtures were being washed with toluene (5 mL) and Et O (2 × 10 mL), then
2
1
13
1
19
1
purchased from BOC Industrial Gases. H, C{ H}, F{ H}, dried in vacuo to yield the desired product.
(4-(Bromomethyl)benzyl)triphenylphosphonium bromide (2a).
using a Bruker AVIII 400 spectrometer. High Resolution This compound was originally synthesised by Wang et al.
3
1
1
P{ H}, COSY, HSQC, and HMBC NMR spectra were obtained
2
8
Electrospray Mass Spectrometry was carried out on a Waters Following general procedure 1, the title compound was pre-
LCT Premier (ES-ToF)/Acquity i-Class spectrometer. HPLC was pared from α,α′-dibromo-p-xylene (0.53 g, 2 mmol) and tri-
performed on an Agilent 1200 Series Liquid Chromatograph phenylphosphine (0.54 g, 2 mmol) as a white solid (0.86 g,
1
with UV and LabLogic Flow-Count detector with a sodium- 82%). H NMR (400 MHz, CDCl
3
) δ
H
(ppm): 7.73 (9H, m,
3
iodide probe (B-FC-3200). Mobile phase A contained water m/p-Ar), 7.60 (6H, m, o-Ar), 7.11 (4H, d, JHH = 1.3 Hz, C
with 0.1% TFA, and mobile phase B contained MeCN with 5.50 (2H, d, J = 14.6 Hz, CH̲ P), 4.37 (2H, d, J
2 HH
6
H
̲
4
),
2
4
= 1.3 Hz,
HP
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Dalton Trans., 2018, 47, 15448–15457 | 15453

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Paper
Dalton Transactions
1
3
1
CH
̲
2
Br). C{ H}-NMR (100 MHz, CDCl
3
) δ
C
(ppm): 138.2 (d,
Triphenyl(4-((4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecan-1-yl)methyl)benzyl)phosphonium bromide
(3a). Following general procedure 2, the title compound was
prepared from DO3Atriester·HBr (0.30 g, 0.5 mmol), 2a (0.26 g,
4
5
2
JCP = 4.4 Hz, p-Ar), 135.1 (d, J = 2.8 Hz), 134.5 (d, J
.3 Hz, o-Ar), 132.1 (d, JCP = 6.1 Hz, C
.1 Hz, m-Ar), 129.5 (d, JCP = 3.5 Hz, C
=
=
=
CP
CP
3
3
9
6
8
̲
6
H
4
), 130.3 (d, JCP
), 127.9 (d, JCP
4
3
̲
6
H
4
1
.8 Hz), 117.9 (d, JCP = 85.9 Hz, i-Ar), 33.0 (C ̲H Br), 30.5 0.5 mmol) and Na CO (0.26 g, 2.5 mmol) as a white solid
2 2 3
1
31
1
1
(d,
J
CP = 47.2 Hz, C
̲
2
P). P{ H}-NMR (162 MHz, CDCl
23BrP tt, JHH = 6.9, JHH = 1.7 Hz, p-Ar), 7.74–7.63 (12H, m, o/m-Ar),
7.05–7.04 (4H, m, C H ), 5.33 (2H, broad s, CH P), 3.64 (2H, s,
3
)
3 H
(0.37 g, 76%). H NMR (400 MHz, CDCl ) δ (ppm): 7.78 (3H,
3
4
δ
P
(ppm): 23.5. HRMS (ES-TOF+): m/z calcd for C26H
+
(
[M − Br] ) 445.0721, found 445.0737.
4-(Bromomethyl)benzyl)tri-p-tolylphosphonium bromide (2b).
Following general procedure 1, the title compound was (18H, s, cis-C(CH
prepared from α,α′-dibromo-p-xylene (0.53 g, 2 mmol) and tri- (100 MHz, CDCl ) δ (ppm): 173.7 (C
̲
̲
6
4
2
(
C
6
H
4
CH
̲
2
), 3.41–2.11 (22H, m, macrocycle H
), 1.43 (9H, s, trans-C(CH
vO), 172.7 (C
CP
CP = 2.7 Hz), 134.4 (d, J =
̲/ CH̲ CO ), 1.47
). C{ H}-NMR
vO), 135.6
3 2
1
3
1
3
)
3
3
)
3
̲
̲
C
5
4
3
p-tolylphosphane (0.72 g, 2 mmol) as a white solid (1.04 g, (d,
2%). H NMR (400 MHz, CDCl
m-Ar), 7.38 (6H, dd, J = 8.2, J = 3.2 Hz, o-Ar), 7.13 (2H, d, (d, JCP = 12.8 Hz), 129.9 (d, JCP = 8.7 Hz), 117.1 (d, JCP
HH = 8.1 Hz, C
), 5.24 (2H, d, JHP = 14.5 Hz, CH
.44 (9H, s, CH
J
CP = 4.0 Hz), 135.3 (d,
J
1
3
4
9
3
) δ
H
(ppm): 7.54 (6H, m, 9.8 Hz), 131.6 (d, JCP = 5.3 Hz), 130.9 (d, JCP = 1.8 Hz), 130.4
3
3
2
2
1
=
HP
HH
3
3
4
J
6
H
̲
4
2
), 7.08 (2H, dd, JHH = 8.4, JHP = 2.4 Hz, 86.0 Hz), 83.1 (cis-C(
̲
CH
3
)
3
), 82.6 (trans-C(
̲
CH
3
)
3
), 57.9, 57.0, 50.9,
1
C
6
H
̲
4
̲
2
P), 4.38 (2H, s, CH
2
Br), 30.8 (d, JCP = 46.5 Hz, C
̲
2
P), 28.2 (C(C
̲
3
)
3
), 28.0 (C(C
̲H
3
)
3
).
1
3
1
31
1
2
1
1
1
1
3
̲
). C{ H}-NMR (100 MHz, CDCl ) δ (ppm):
P{ H}-NMR (162 MHz, CDCl ) δ (ppm): 23.1. HRMS
3
3
C
3
P
5
4
+
46.3 (d, JCP = 2.6, Hz, C
̲
6
H
4
), 138.1 (d, JCP = 3.9 Hz, o-Ar), (ES-TOF+): m/z calcd for C52
H
72
N
4
O
6
P ([M − Br] ) 879.5190,
P ([M − Br
6H4), + 2H] ) 293.8443. Found: 879.5258, 440.2600, 293.8420.
), 114.6 (d, JCP = 88.7 Hz, i-Ar), Tri-p-tolyl(4-((4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-
CP = 48.1 Hz, C P), 22.0 (C ). tetraazacyclododecan-1-yl)methyl)benzyl)phosphonium bromide
P{ H}-NMR (162 MHz, CDCl ) δ (ppm): 22.4. HRMS (3b). Following general procedure 2, the title compound was
2
3
2+
34.3 (d, JCP = 10.4 Hz, p-Ar), 132.0 (d, JCP = 5.7 Hz, C
6
H
4
),
C
52
H
73
N
3+
4
O
6
P ([M − Br + H] ) 440.2628, C52
H
74
N
4
O
6
3
4
30.4 (d, J = 3.4 Hz, m-Ar), 129.5 (d, J = 3.6 Hz, C
̲
CP
CP
2
1
6 4
28.1 (d, JCP = 8.6 Hz, C̲ H
1
3.1 (C
̲
H
2
Br), 30.9 (d,
J
̲
2
̲H
3
3
1
1
3
P
+
(
5
ES-TOF+): m/z calcd for C29
29.1669. Anal. calcd for C29
H 5.14 (5.26).
H
H
29BrP ([M − Br] ) 529.1660, found prepared from DO3Atriester·HBr (0.30 g, 0.5 mmol), 2b (0.28 g,
29PBr (found): C, 61.29 (61.22); 0.5 mmol) and Na CO (0.26 g, 2.5 mmol) as a white solid
(0.23 g, 47%). H NMR (400 MHz, CDCl ) δ (ppm): 7.57–7.52
2
2
3
1
3
H
3
4
(
4-(Bromomethyl)benzyl)tris(3,5-dimethylphenyl)phosphonium (6H, m, o-Ar), 7.43 (6H, dd, JHH = 8.3, JHH = 3.3 Hz, m-Ar),
bromide (2c). Following general procedure 1, the title com- 7.07 (2H, dd,
pound was prepared from α,α′-dibromo-p-xylene (0.53 g,
3
4
J
HH = 8.3,
J
HH = 2.3 Hz, C
6
H
̲
4
), 7.03 (2H, d,
P), 3.68
3
2
JHH = 8.1 Hz, C H
̲
), 5.18 (2H, d, J = 14.4 Hz, CH̲
), 3.43–2.18 (16H, m, macrocycle H
), 2.47 (9H, s, ArCH ) 1.48 (18H, s, cis-C
6
4
HP
2
2
2
mmol) and tris(3,5-dimethylphenyl)phosphane (0.87 g, (2H, s, C
6
H
4
CH
mmol) as a white solid (2.74 g, 88%). H NMR (400 MHz, (6H, m, CH CO
) ), 1.43 (9H, s, trans-C(CH̲ ) ). C{ H}-NMR (100 MHz,
3 3
̲
2
̲) 3.04–3.00
1
̲
2
2
3
3
CDCl ) δ (ppm): 7.34 (3H, s, p-Ar), 7.21 (6H, d, J = 13.0 Hz, (CH
̲
3
H
HP
3 3
3
3
o-Ar), 7.15 (2H, d, JHH = 7.6 Hz, C
.0, JHP = 2.7 Hz, C
.40 (2H, s, CH Br), 2.36 (18H, s, CH
100 MHz, CDCl
38.0 (d, JCP = 3.5 Hz, C
32.1 (d, J = 5.1 Hz, C
29.3 (d, JCP = 3.5 Hz, p-Ar), 128.5 (d, JCP = 8.7 Hz, m-Ar), 31.1 (d,
17.8 (d,
6
H
4
), 7.02 (2H, dd, JHH
=
CDCl ),
P), 135.6 (C
C{ H}-NMR H ), 131.0 (d, J = 12.5 Hz, C̲ H ), 130.8 (m-Ar), 127.2 (d,
3
) δ
C
(ppm): 173.7 (C
̲
̲
6
H
4
4
2
2
4
8
4
(
1
1
1
1
4
6
H
4
), 5.21 (2H, d, JHP = 14.3 Hz, CH
2
6 4
̲ H
1
3
1
3
̲
̲
).
C
̲
2
3
6
4
CP
6
4
2
4
1
3
) δ
C
(ppm): 140.3 (d, JCP = 12.3 Hz, C
6
H
H
4
),
J
CP = 8.5 Hz, p-Ar), 114.6 (d,
(CH ), 82.6 (C(CH ), 58.0 (C
CO ), 51.0 (broad, macrocycle C)
J
CP = 88.7 Hz, i-Ar), 83.0
), 56.9 (C CO ), 56.0
, 49.4 (broad, macrocycle C)
), 28.0 (trans-C(C ),
). P{ H}-NMR (162 MHz, CDCl ) δ (ppm): 22.1.
5
4
6
H
4
), 136.8 (d, JCP = 3.5 Hz, C
6
4
), (C
̲
3
)
3
̲
3
)
3
6
H
4
C
̲
2
̲
2
2
3
2
̲
H ), 131.7 (d, J = 10.1 Hz, o-Ar), (CH
4
̲
̲
̲ ,
CP
CP
2
2
4
3
1
J
CP, CH
̲
2
P), 28.2 (cis-C(C
̲
3
)
3 3
̲H )
1
1
31
1
J
̲
CP = 84.5 Hz, i-Ar), 33.1 (C
H P), 21.5 (CH
2
̲
2
Br), 30.9 (d,
J
CP
=
22.0 (Ar-C
̲
3
3
P
3
1
1
+
6.6 Hz, C
̲
). P{ H}-NMR (162 MHz, CDCl ) HRMS (ES-TOF+): m/z calcd for C H N O P ([M − Br] )
3
3
55 78 4 6
2
+
δ
P
(ppm): 22.7. HRMS (ES-TOF+): m/z calcd for C32
− Br] ) 529.1660, found 529.1643. Anal. calcd for 921.5684, 461.2828.
H
79 4 6
35BrP 921.5659, C55H N O P ([M − Br + H] ) 461.2863. Found:
+
([M
C H PBr (found): C, 62.97 (62.98); H, 5.78 (5.73).
Tris(3,5-dimethylphenyl)(4-((4,7,10-tris(2-(tert-butoxy)-2-
oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)phos-
phonium bromide (3c). Following general procedure 2, the title
compound was prepared from DO3Atriester·HBr (0.30 g,
3
2
35
2
General procedure 2 – coupling of triarylphosphane-
substituted bromoxylene to DO3Atriester hydrobromide (3)
The methodology was a modification of the procedure 0.5 mmol), 2c (0.31 g, 0.5 mmol) and Na
2
CO
3
(0.26 g,
3
7
1
described by Kardashinsky et al.
To a solution of 2.5 mmol) as a white solid (0.36 g, 70%). H NMR (400 MHz,
3
DO3Atriester·HBr (1 eq.) and triarylphosphane-substituted bro- CDCl ) δH (ppm): 7.35 (3H, s, p-Ar), 7.21 (6H, d, J = 13.1 Hz,
3
HP
3
3
moxylene (1 eq.) in MeCN (15 mL), Na
and the resulting suspension was heated to reflux and stirred
2
CO
3
(5 eq.) was added, o-Ar), 7.11 (2H, d,
J
HH = 8.0 Hz), 7.05 (2H, dd,
J
HH = 8.2,
P), 3.68 (2H, s),
),
), 1.44 (9H, s,
). C{ H}-NMR (100 MHz, CDCl ) δ (ppm):
vO), 140.2 (d, JCP = 12.6 Hz, C H4),
4
2
JHP = 2.4 Hz), 5.15 (2H, d, JHP = 14.2 Hz, CH̲
2
under an N2 atmosphere overnight. The inorganic base was 3.05 (2H, s), 3.01 (4H, s), 2.97–2.16 (16H, m, macrocycle H
removed by filtration and the solvent removed under reduced 2.36 (18H, s, Ar–CH ), 1.48 (18H, s, cis-C(CH
pressure. The resulting residue was recrystallized from trans-C(CH
acetone/Et O to yield the desired product. 173.6 (CvO), 172.7 (C
̲
̲
3
3 3
̲ )
1
3
1
3
)
3
3
C
2
̲
̲
̲
2
6
15454 | Dalton Trans., 2018, 47, 15448–15457
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Dalton Transactions
Paper
+
2+
1
36.8 (p-Ar), 135.9 (C
̲
6
H
4
), 131.6 (o-Ar), 131.5 (C
̲
6
H
4
), 130.7 ([M − CF
(C̲ H ), 127.5 (d, J = 8.3 Hz, m-Ar), 117.7 (d, J = 84.9 Hz, 377.1924. Found: 753.3786, 377.2102.
6
3
CO
2
] ) 753.3781, C43
H
55
N
4
O
6
3 2
P ([M − CF CO + H] )
3
1
4
CP
CP
i-Ar), 82.9 (C
̲H
2
CO
), 55.9 (C
broad, macrocycle C
), 27.9 (trans-C(C
162 MHz, CDCl ) δ (ppm): 22.4. HRMS (ES-TOF+): m/z calcd (2H, d, JHH = 7.7 Hz, C
for C H N O P ([M − Br] ) 963.6129, C H N O P ([M − Br + 7.00 (2H, d, J
2
), 82.5 (C
CO ), 50.8 (broad, macrocycle C
), 30.9 (d, J = 48.5 Hz, C
̲
2
CO
2
), 57.9 (C
6
H
4
C̲
2
), 56.7
), 49.2 the title compound was prepared from 3c (0.33 g, 0.32 mmol)
̲H P), 28.1 (cis- and trifluoroacetic acid (3.3 mL) as a white solid (0.17 g, 57%).
3
DO3A-xy-TXP trifluoroacetate. Following general procedure 3,
(
(
C
̲
H
2
CO
2
̲
H
2
2
̲
1
̲
CP
2
1
1
1
C(C
(
̲H
3
)
3
̲
3
)
3
), 21.5 (Ar-C
̲
3
). P{ H}-NMR
H
H NMR (400 MHz, MeOD) δ (ppm): 7.52 (3H, s, p-Ar), 7.49
3
3
3
P
6 4
H̲ ), 7.18 (6H, d, JHP = 13.1 Hz, o-Ar),
= 6.7 Hz, C H̲ ), 4.68 (2H, d, J = 15.0 Hz,
4 HP
+
3
2
58
84
4
6
58 85
4
6
HH
6
2
+
H] ) 482.3098. Found: 963.6139, 482.2990.
CH
m, macrocycle H
MeOD) δC (ppm): 161.6 (C
2
̲ P), 4.24 (2H, s), 3.83 (2H, s), 3.64 (2H, s), 3.42–3.01 (18H,
1
3
1
̲
3
). C{ H}-NMR (100 MHz,
2
̲
̲
=
=
CP
General procedure 3 – deprotection of tert-butyl protected
DO3A-xy-TAP compounds using trifluoroacetic acid (4)
4
4
1
5
3.7 Hz, C
.5 Hz, C
H ), 117.5 (d, J = 84.7 Hz, i-Ar), 115.4 (m-Ar), 56.8,
̲
2
̲ H ), 131.5 (C̲ H ), 131.2 (d, JCP = 10.4 Hz, o-Ar),
6 4 6 4
1
The methodology was a modification of the procedure 118.3 (C
̲
6
4
CP
3
7
described by Kardashinsky et al. tert-butyl protected com- 54.4, 49.8 (broad s), 49.1 (broad s), 48.7 (broad s), 29.2 (d,
1
19
1
pounds were dissolved in trifluoroacetic acid (reaction mixture
J
CP = 49.7 Hz, C
2 3
̲H P), 19.9 (C ̲H ). F{ H}-NMR (377 MHz,
−1
31
1
concentration 0.1 g mL ) and stirred at rt under an N atmo- MeOD) δF (ppm): −72.6. P{ H}-NMR (162 MHz, MeOD)
2
sphere for 24 h. The acid was removed in vacuo, before the
residue was taken up in H O (50 mL) and washed with CHCl
3 × 10 mL). The aqueous solvent was removed under reduced 398.2159. Found: 795.4326, 398.2164.
P 60 4 6
δ (ppm): 22.3. HRMS (ES-TOF+): m/z calcd for C46H N O P
+
2+
2
3
([M − CF
3
CO
2
] ) 795.4250, C46
H
61
N
4
3 2
OP ([M − CF CO + H] )
(
pressure, before the residue was purified via semi-preparative
HPLC methods.
DO3A-xy-TPP trifluoroacetate. Following general procedure
Radiochemistry procedures
6
8
General synthesis of [ Ga]Ga-DO3A-xy-TAP. The method-
3
, the title compound was prepared from 3a (0.25 g, ology was a modification of the procedure described by
2
8
0
.26 mmol) and trifluoroacetic acid (2.5 mL) as a white solid Wang et al. To a solution of DO3A-xy-TAP trifluoroacetate
1
(0.16 g, 73%). H NMR (400 MHz, MeOD) δ (ppm): 7.91 (3H, (approx. 100 μg) in NH OAc buffer (0.1 M, 0.5 mL), an aliquot
H
4
3
4
68
td, JHH = 7.3, JHH = 1.8 Hz, p-Ar), 7.78–7.62 (12H, m, o,m-Ar), of [ Ga]GaCl
3
in 0.1 M HCl (approx. 150 MBq, 0.5 mL) was
HH = 8.0, added, and the resulting mixture was heated at 100 °C for
P), 4.34 30 min to yield the desired product. Initial QC was performed
3
3
7
.51 (2H, d,
J
HH = 7.8 Hz, C
JHP = 2.7 Hz, C H ), 4.95 (2H, d, J = 15.1 Hz, CH
2
6
H̲
), 7.08 (2H, dd,
J
4
2
̲
̲
6
4
HP
(
(
(
2H, s), 3.97 (2H, s), 3.64 (2H, s), 3.39 (4H, s), 3.29–3.02 using iTLC and a mobile phase of 0.1 M disodium EDTA and
16H, m). C{ H}-NMR (100 MHz, MeOD) δ
C
1
3
1
C
(ppm): 162.8 by the analytical HPLC method described previously.
vO), 136.6 (p-Ar), 135.3 ( J = 9.1 Hz, m-Ar), Determination of Log D values. As an index for lipophilicity,
), 131.5 ( JCP = 13.6 Hz, o-Ar), 119.6 octanol/phosphate buffered saline (PBS) partition coefficient
3
̲
vO), 162.5 (C
̲
CP
3
2
1
33.2 ( JCP = 5.2 Hz, C
6
H
4
1
68
(C
6
H
4
), 119.0 ( JCP = 86.9 Hz, i-Ar), 116.7 (C
6
5
5.6, 54.1, 50.5 (broad s), 50.4 (broad s), 30.3 ( J = 48.6 Hz, determined. A general methodology is as follows: aliquots of
CP
1
9
1
C
̲H
2
1
P). F{ H}-NMR (377 MHz, MeOD) δ
F
(ppm): −73.0. the radiolabelled compound (10 µL, ∼4 kBq) were added to
3
1
P{ H}-NMR (162 MHz, MeOD) δ
P
(ppm): 26.8. HRMS vials containing a layered mixture of n-octanol (500 µL) and
+
(ES-TOF+): m/z calcd for C H N O P ([M − CF CO ] ) PBS solution (pH 7.4, 500 µL). After vortex mixing for 1 min,
4
0
48
4
6
3
2
2
+
7
11.3311, C40
H
49
N
4
O
6
P ([M − CF
3
CO
2
+ H] ) 356.1689. Found: the mixture was centrifuged to separate the octanol–PBS
4
7
11.3320, 356.1727.
phases (10 rpm, 2 min), before aliquots of the two phases
DO3A-xy-TTP trifluoroacetate. Following general procedure 3, (100 µL) were taken, and transferred into vials for counting.
the title compound was prepared from 3b (0.21 g, 0.23 mmol) Relative amounts of test compound in each phase were deter-
and trifluoroacetic acid (2.1 mL) as a white solid (0.10 g, 57%). mined by an automatic gamma counter. The log D value was
1
H-NMR (400 MHz, MeOD) δH (ppm): 7.58–7.52 (12H, m, reported as the average of data obtained in 6 independent
3
o/m-Ar), 7.47 (2H, d, JHH = 8.2 Hz, C
C H̲ ), 4.83 (2H, d, JHP = 15.3 Hz, CH̲
6 4 2
4
6
H
̲
4
), 7.09 (2H, broad s, experiments.
P), 4.48 (2H, broad s),
.15 (2H, broad s), 3.78–3.43 (4H, m, CH2CO H), 3.29–2.90
2
In vitro tumour uptake study
). C{ H}-NMR For the tumour uptake study, MDA-MB 231 breast cancer cells
̲
2
1
3
1
(16H, m, macrocycle H
̲
̲
3
6
8
68
(100 MHz, MeOD) δ
C
(ppm): 174.2 (C
̲
̲
3
4
(
C
p-Ar), 135.2 (d, J = 10.3 Hz, C̲ H ), 133.2 (d, J = 5.4 Hz, solved in serum-free cell medium to give a concentration of
CP 6 4 CP
2
−1
6
H
4
), 132.0 (d,
J
CP = 12.4 Hz, o-Ar), 121.9 (C
̲
6
H
4
), 119.0 1 MBq mL . 100 kBq aliquots of radiotracer were added to
1
5
(
m-Ar), 115.7 (d, JCP = 90.2 Hz, i-Ar), 113.2 (C
6
H
4
), 58.6, 55.4, cell samples (2 mL, 5 × 10 cells per mL) and incubated for
1
5
4
δ
̲
H CO H), 50.8 (broad, macrocycle C
̲
=
30 min. The cell supernatant was transferred to tubes for
2
2
1
9
1
8.7 Hz, C ̲H P), 21.8 (C ̲H
2 3
3
1
1
F
P
2
4
6
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serum-free cell medium (700 μL). The supernatant and the
harvested cells were assayed in an automatic gamma counter
Acknowledgements
to determine percentage tracer uptake. The experiments were We would like to thank the KCL/ICL EPSRC CDT in Medical
performed in triplicate.
Imaging (grant EP/L015226/1) for providing funding for
A. S. and P. G. This research was supported by the Centre of
Excellence in Medical Engineering funded by the Wellcome
Trust and EPSRC (203148/Z/16/Z).
Langendorff isolated heart perfusion
Animals. Male Wistar rats (240–275 g; Charles River
Laboratories) were used for all experiments. Animal pro-
cedures were in accordance with the Animals (Scientific
Procedures) Act, UK, 1986.
Notes and references
The triple-γ-detector system. We developed a system for
characterizing the pharmacokinetics of radionuclide passage
through an isolated perfused heart comprising 3 orthogonally
orientated lead-collimated Na/I γ detectors positioned: (i) 3 cm
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5 cm upstream of the heart cannula (to provide a radiotracer
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Each was connected to a modified GinaSTAR instant thin-layer
chromatography system running Gina software (Raytest Ltd).
Experimental protocol. Rats were anaesthetized with sodium
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00 IU kg− respectively, i.p.) and their hearts were excised and
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modified Krebs–Henseleit buffer (mKHB) containing NaCl
4 3
(118 mM), KCl (5.9 mM), MgSO (2.3 mM), NaHCO (25 mM),
EDTA (0.65 mM), glucose (11.1 mM) and CaCl (2.5 mM). The
2
mKHB was freshly prepared and filtered before use, and was
gassed with 95% O and 5% CO and used at 37 °C. A left ven-
2 2
tricular balloon was used to measure contractile function.
After a 10 min stabilization period, a bolus of radiotracer
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−
1
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7 °C was infused via the arterial line at 1.4 mL min , whilst
the flow rate at the peristaltic pump was reduced by 10%.
A further 1 MBq radiotracer bolus was administered 5 minutes
after the commencement of CCCP infusion. The radiotracer
retained after 15 min in both the healthy and CCCP-infused
hearts was reported as the average of the normalized activity
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Author contributions
1
N.L. and R.S. jointly supervised the work. A.S. carried out the
synthetic chemistry, radiochemistry and isolated heart per-
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Conflicts of interest
There are no conflicts to declare.
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