Orthogonal 18F and 64Cu labelling of functionalised bis(thiosemicarbazonato) complexes

Article abstract of DOI:10.1039/b926980k

The synthesis of three pairs of orthogonally labelled fluorinated Cu bis(thiosemicarbazonato) complexes is presented. These are the first examples of ¹⁸F-labelled Cu(ii)-complexes designed to serve as new hypoxia selective PET tracers and as mechanistic probes to study the mode of action of this class of markers. In vitro evaluation revealed that the fluorinated Cu-complex derived from amide coupling is suitable for in vivo work.

Full text of DOI:10.1039/b926980k

Chemical Communications
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Volume 46 | Number 23 | 21 June 2010 | Pages 4001–4204
ISSN 1359-7345
COMMUNICATION
FEATURE ARTICLE
Véronique Gouverneur et al.
Karl J. Hale et al.
Orthogonal 18F and 64Cu
labelling of functionalised
bis(thiosemicarbazonato) complexes
Total synthesis of powerful new
inhibitors of β-catenin/TCF4- and
E2F-mediated gene transcription
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18 64
Orthogonal F and Cu labelling of functionalised
bis(thiosemicarbazonato) complexesw
Laurence Carroll,a Romain Bejot,a Rebekka Hueting,a Robert King,a Paul Bonnitcha,a
a
a
a
b
c
ˆ
Simon Bayly, Martin Christlieb, Jonathan R. Dilworth, Antony D. Gee, Jerome Declerck
´
a
´
and Veronique Gouverneur*
Received 23rd December 2009, Accepted 24th March 2010
First published as an Advance Article on the web 20th April 2010
DOI: 10.1039/b926980k
The synthesis of three pairs of orthogonally labelled fluorinated
Cu bis(thiosemicarbazonato) complexes is presented. These are
the first examples of 18F-labelled Cu(II)-complexes designed to
serve as new hypoxia selective PET tracers and as mechanistic
probes to study the mode of action of this class of markers.
In vitro evaluation revealed that the fluorinated Cu-complex
derived from amide coupling is suitable for in vivo work.
either at the metal with 64Cu or at the ligand with 18F
(Scheme 1). Careful SAR studies conducted by Lewis et al.
and Kelly et al. indicate that there appears to be strong
correlation between the redox potential of the copper complex
and its hypoxia selectivity.11,12 Typically, the redox potential is
controlled primarily by the substitution pattern of the carbon
backbone, whereas modification of the exocyclic amino
group modifies lipophilicity and biodistribution.13–16 These
observations combined with synthetic considerations suggest
that manipulation of the exocyclic amino group is more
suitable for 18F-labelling. We opted for a synthetic route based
on the use of 18F-labelled prosthetic groups as this modular
approach should comply with the challenges associated with
the handling of short half-life 18F (109.7 min) and allow for
easier structural variation.
Low oxygen level in cells, known as hypoxia, has direct
implications for the treatment of various diseases including
cancer.1,2 Hypoxic tumours are typically more difficult to
eradicate with a lower survival rate for patients not diagnosed
correctly.3 It is therefore critical to identify hypoxic
tumours from the outset, so that treatment can be personalised.
A
commonly used non-invasive diagnostic method to
detect tumours is positron emission tomography (PET).4–6
Nitroimidazole-based tracers such as [18F]fluoromisonidazole
([18F]FMISO), and metal complexes, for example diacetyl-di-
Three reactions were selected based on the availability of
well-established protocols to access the necessary 18F-labelled
component: an imine condensation with [18F]4-fluorobenz-
aldehyde, an amide bond formation with [18F]4-fluorobenzoic
acid and a Huisgen 1,3-dipolar cycloaddition with [18F]2-
fluoro-1-azidoethane. Both imine and amide bond formation
reactions were reported in the literature to access various
functionalized bis(thiosemicarbazonato)copper(II) complexes
metal radiolabelled.16 In contrast, click chemistry is unprecedented
for copper radiopharmaceutical synthesis despite its extensive
use in the context of chemical biology and more recently in
radiochemistry.17–20 This is likely due to possible complication
(N4-methyl-thiosemicarbazonato)-[64Cu]copper(II)
([64Cu]-
Cu-ATSM) have been developed to target and image hypoxic
tumours. These metal complexes are labelled at the metal.7–10
Recently, we have questioned whether complexes structurally
related to Cu(II)-ATSM but 18F-labelled at the ligand, might
serve as new hypoxia selective radiotracers for PET. The wide
availability of 18F would open the possibility to use these new
compounds for hypoxia imaging at a large number of clinical
sites worldwide. In addition, these new 18F-labelled metal
complexes, structurally related to Cu(II)-ATSM, are unique
probes to bring new insight into the mode of action of this
class of hypoxia selective radiotracers by examining the
metabolic fate of the copper complex in vivo. Valuable
information on the mechanism of these new complexes would
be gained by examining the biodistribution profile of both the
ligand and the metal. This can be achieved if these two
entities could be radiolabelled independently. This so-called
‘‘orthogonal labelling’’ strategy requires the preparation of a
pair of structurally identical Cu-ATSM derivatives labelled
a University of Oxford, Chemistry Research Laboratory,
Mansfield Road, Oxford, UK.
E-mail: veronique.gouverneur@chem.ox.ac.uk;
Tel: +44 01865275622
b GSK Clinical Imaging Centre, Imperial College London,
Hammersmith Hospital, Du Cane Road, London, UK
c Siemens Molecular Imaging, 23–38 Hythe Bridge Street, Oxford, UK
w Electronic supplementary information (ESI) available: Full
characterisation of novel compounds and HPLC data of radiolabelling
experiments. See DOI: 10.1039/b926980k
Scheme
1 Orthogonal labelling: concept and retrosynthetic
approach.
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arising from the propensity of proligands (or zinc complexes)
to metallate (or transmetallate) in the presence of Cu2+ ions,
which are typically used to promote 1,3-dipolar cycloadditions
of azides with alkynes. This strategy will therefore require
careful synthetic planning.
Initial work began by studying the coupling of Zn-ATSM/A
with 4-fluorobenzaldehyde 2.13 Imine condensation
1
performed in acetonitrile at 80 1C delivered an inseparable
E/Z mixture (1 : 4) of hydrazone 3 in 56% yield. Transmetallation
with [64Cu]Cu(OAc)2 was quantitative within 5 min to give
the 64Cu-complex 4 radiochemically pure as judged by radio-
HPLC. For the preparation of the novel orthogonally labelled
[18F]4, [18F]4-fluorobenzaldehyde [18F]2 was firstly prepared
in acetonitrile by nucleophilic aromatic substitution of
4-trimethylammoniumbenzene triflate with [18F]ꢁ/K+-Kryptofix
222.21 Condensation of [18F]2 with Zn-ATSM/A 1 led to the
formation of [18F]3 (E/Z ratio = 5 : 1) in 94% RCY.
Subsequent transmetallation of [18F]3 with Cu(OAc)2 gave
the desired complex [18F]4. All radiochemical yields are
reported decay-corrected. The identity of [18F]2, [18F]4 and
labelled [64Cu]4 was unambiguously confirmed by comparison
of their radio-HPLC trace with the UV-HPLC trace of
independently prepared and fully characterized non-radioactive
material (Scheme 2).
Scheme 3 Synthesis of complex 6.
(‘‘click reaction’’) for the synthesis of copper-based tracers
(Scheme 4). Following a recent literature procedure,22 [18F]9
was prepared by nucleophilic fluorodetagging of the
corresponding fluorous sulfonate and subsequent facile
purification by fluorous solid phase extraction (FSPE).
The Zn-complex 11 was prepared by imine condensation of
Zn-ATSM/A 1 with aldehyde 12.
4-Fluorobenzoic acid 5 was selected to validate the amide
coupling strategy. To access the novel amide complex 6
radiolabelled at the metal, H2ATSM/A 8 was coupled with
4-fluorobenzoic acid in the presence of O-benzotriazole-
N,N,N0,N0-tetramethyluronium hexafluorophosphate (BOP
PF6ꢁ) and N,N-diisopropylethylamine. Addition of [64Cu]-
Cu(OAc)2 to the resulting proligand 7 led quantitatively to
[64Cu]6. Orthogonal 18F-labelling required access to [18F]5,
which was made available within 30 min upon fluorination of
ethyl 4-trimethylammoniumbenzoate triflate with [18F]ꢁ/K+-
Kryptofix 222 followed by hydrolysis. Amide coupling of
[18F]5 with 8 followed by metallation using Cu(OAc)2 gave
[18F]6 in an overall RCY of 32%. The synthesis of [18F]6 from
[18F]5 took less than 20 min (Scheme 3).
Pleasingly, upon treatment with CuSO4 and Na-ascorbate,
the 1,3-dipolar cycloaddition with [18F]-2-fluoro-1-azido-
ethane was successful in DMSO at 100 oC within 15 min.
Subsequent addition of Cu(OAc)2 led to [18F]13 with a RCY
of 84%. The synthesis of [64Cu]13 was uneventful following a
strategy reversing the order of the synthetic steps, the click
coupling between aldehyde 12 and 2-fluoro-1-azidoethane
being carried out prior to imine condensation with
Zn-ATSM/A 1. The resulting Zn-complex was transmetallated
under standardised conditions leading to [64Cu]13 in high
radiochemical yield and purity.
To compare the electrochemical properties of the new
complexes with the parent compound Cu-ATSM, the Cu(II/I)
redox couple was measured for complexes 4, 13 and 6 giving
values of ꢁ0.60 V, ꢁ0.64 V and ꢁ0.62V vs. SCE respectively
[18F]-2-Fluoro-1-azidoethane 9 was considered next to
investigate the value of Huisgen 1,3-dipolar cycloadditions
Scheme 2 Synthesis of complex 4.
Scheme 4 Synthesis of complex 13.
ꢀc
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Table 1 Cell uptake studies
to functionalize bis(thio-semicarbazonato) complexes.
Known methodologies were improved for compatibility with
18F-labelling (t1/2 = 109.7 min). In vitro data indicate that the
fluorinated complex 6 is suitable for in vivo investigation.
Current work is underway to determine whether 18F-labelled
Cu(II) bis(thiosemicarbazonato) complexes can be used as
PET imaging tracers to measure hypoxia and to enhance
our understanding of the mode of action of this family of
markers.
21% (normoxic)
0% (hypoxic)
[
[
[
[
[
[
[
64Cu]Cu-ATSMa,24
64Cu]Cu-ATSMb
64Cu]Cu-ATSMc
64Cu]4b
10%
65%
21.3 ꢂ 0.3%
30.3 ꢂ 2.4%
53.1 ꢂ 1.5%
53.8 ꢂ 2.3%
82.6 ꢂ 1.1%
38.6 ꢂ 0.4%
47.6 ꢂ 0.9%
41.1 ꢂ 2.2%
48.8 ꢂ 1.3%
63.5 ꢂ 0.9%
91.5 ꢂ 0.1%
35.6 ꢂ 3.4%
64Cu]6c
64Cu]13a
64Cu]13c
a
b
EMT6 suspension assay, 1 h. HT1080 suspension assay, 1 h.
HT1080 adherent, 3 h.
c
Notes and references
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(for Cu(II)-ATSM: ꢁ0.62 V vs. SCE).23 These data indicate
that all new complexes display an electrochemical behavior
similar to Cu(II)-ATSM. While the relationship between
lipophilicity and cellular uptake is expected to be complex,
this property is critically important in the context of bio-
distribution. With a log P value of 1.50, Cu(II)-ATSM is a
suitable tracer despite its relatively high liver uptake.11 Log P
values of 1.55, 1.70 and 2.50 were measured for complexes 6,
13 and 4, respectively. These data indicate that 6 and 13
emerge as the closest Cu(II)-ATSM analogues since their log
P values are similar. With a higher log P value of 2.50,
complex 4 might sequester itself within membranes becoming
inaccessible to intracellular reducing agents within the cytosol,
and therefore cannot be elected a suitable candidate for in vivo
experimentation.
6 G. J. R. Cook, S. Houston, S. F. Barrington and I. Fogelman,
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Available from: http://micad.nih.gov.
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Hypoxia selectivity can be measured in vitro by oxygen
dependent cellular uptake measurements (See SI for detailsw).
Hypoxia selectivity of the fluorinated complexes 4, 6 and 13
was tested and compared with [64Cu]Cu-ATSM (Table 1).
Using the HT1080 suspension assay, [64Cu]4 showed
approximately 10% higher uptake in hypoxic cells compared
to normoxic cells after 10 min, however after 1 h, uptake under
normoxia had become higher than that under hypoxia. Data
for [64Cu]6 were much more encouraging, and in the HT1080
adherent assay it showed excellent hypoxia selectivity with
64% uptake after 3 h in hypoxic cells compared to 54% in
normoxic cells. The 10% difference is equivalent to that
observed for [64Cu]Cu-ATSM, although uptake of [64Cu]6
was higher in comparison at both oxygen concentrations.
Although a preliminary EMT6 suspension assay on [64Cu]13
showed some selectivity for hypoxic cells (but of lower magnitude
than [64Cu]Cu-ATSM), in the adherent assay with HT1080
cells no significant difference in uptake of the complex under
normoxic and anoxic conditions was observed. From these
results [64Cu]6 was earmarked for future in vivo study.
12 C. Kelly, M. Kelly, R. Bejot, S. Bayly, Q. Guo, J. Dilworth,
V. Gouverneur, M. Brady and J. Declerck, J. Nucl. Med., 2008, 49,
122P.
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In conclusion, the fluorinated bis(thiosemicarbazone)
complexes 4, 6 and 13 were prepared and radiolabelling with
18F or 64Cu led to six new radiotracers. These are the first
examples of 18F-labelled diacetyl-di(N4-methyl-thiosemi-
carbazonato)-copper(II) derivatives. It has been demonstrated
for the first time that ‘click’ chemistry is a viable transformation
ꢀc
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4054 | Chem. Commun., 2010, 46, 4052–4054