Towards dual SPECT/optical bioimaging with a mitochondrial targeting, 99mTc(i) radiolabelled 1,8-naphthalimide conjugate

Article abstract of DOI:10.1039/c9dt04024b

A series of six different 1,8-naphthalimide conjugated dipicolylamine ligands (L^1-6) have been synthesised and characterised. The ligands possess a range of different linker units between the napthalimide fluorophore and dipcolylamine chelator which allow the overall lipophilicity to be tuned. A corresponding series of Re(i) complexes have been synthesised of the form fac-[Re(CO)₃(L^1-6)]BF₄. The absorption and luminescence properties of the ligands and Re(i) complexes were dominated by the intramolecular charge transfer character of the substituted fluorophore (typically absorption ca. 425 nm and emission ca. 520 nm). Photophysical assessments show that some of the variants are moderately bright. Radiolabelling experiments using a water soluble ligand variant (L⁵) were successfully undertaken and optimised with fac-[^99mTc(CO)₃(H₂O)₃]⁺. Confocal fluorescence microscopy showed that fac-[Re(CO)₃(L⁵)]⁺ localises in the mitochondria of MCF-7 cells. SPECT/CT imaging experiments on na?ve mice showed that fac-[^99mTc(CO)₃(L⁵)]⁺ has a relatively high stability in vivo but did not show any cardiac uptake, demonstrating rapid clearance, predominantly via the biliary system along with a moderate amount cleared renally.

Full text of DOI:10.1039/c9dt04024b

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Towards dual SPECT/optical bioimaging with a
99m
mitochondrial targeting,
Tc(I) radiolabelled
Cite this: Dalton Trans., 2020, 49, 511
1,8-naphthalimide conjugate†
a
b
b
b
Adam H. Day, Juozas Domarkas, Shubhanchi Nigam, Isaline Renard,
b
b
c
c
Christopher Cawthorne,
Ian A. Fallis, * Stephen J. Archibald * and Simon J. A. Pope
Benjamin P. Burke, Gurmit S. Bahra, Petra C. F. Oyston,
a
b
a
*
1
–6
A series of six different 1,8-naphthalimide conjugated dipicolylamine ligands (L ) have been synthesised
and characterised. The ligands possess a range of different linker units between the napthalimide fluoro-
phore and dipcolylamine chelator which allow the overall lipophilicity to be tuned. A corresponding series
1
–6
3 4
of Re(I) complexes have been synthesised of the form fac-[Re(CO) (L )]BF . The absorption and lumine-
scence properties of the ligands and Re(I) complexes were dominated by the intramolecular charge trans-
fer character of the substituted fluorophore (typically absorption ca. 425 nm and emission ca. 520 nm).
Photophysical assessments show that some of the variants are moderately bright. Radiolabelling experi-
5
ments using a water soluble ligand variant (L ) were successfully undertaken and optimised with fac-
9
9m
+
5 +
Received 14th October 2019,
Accepted 9th December 2019
[
3 2 3 3
Tc(CO) (H O) ] . Confocal fluorescence microscopy showed that fac-[Re(CO) (L )] localises in the
99m
mitochondria of MCF-7 cells. SPECT/CT imaging experiments on naïve mice showed that fac-[
5 +
Tc
DOI: 10.1039/c9dt04024b
(
CO) (L )] has a relatively high stability in vivo but did not show any cardiac uptake, demonstrating rapid
3
rsc.li/dalton
clearance, predominantly via the biliary system along with a moderate amount cleared renally.
3
useful imaging agents. Combined single molecule SPECT/
fluorescence agents can provide early phase high sensitivity
Introduction
The different bioimaging modalities each have intrinsic imaging of in vivo biodistribution using SPECT, followed by
4
strengths and weaknesses, with no single clinically used tech- longitudinal optical imaging. Additionally, PET or SPECT
nique providing comprehensive information. Prospective imaging can be used to identify diseased tissue non-invasively,
multimodal imaging agents are designed to combine the which can then be accurately biopsied or surgically resected
5
–7
strengths of individual modalities, while offering complemen- based upon the fluorescence imaging signal.
9
9m
tary or additional clinically useful information not provided by
The clinical use of
Tc in SPECT imaging has been well
1
,2
99m
a single technique. Single photon emission computed tom- established for many decades.
Tc (t1/2 = 6.01 h, γ = 142.7
ography (SPECT) is a high sensitivity nuclear imaging tech- keV) is a gamma emitting radioactive isotope which is used in
nique using short half-life gamma emitting radioisotopes that >80% of clinical nuclear diagnostic imaging scans, with over
8
can image the whole body. In comparison, optical fluorescence 40 million SPECT scans carried out per year worldwide. What
imaging can utilise agents that emit visible or near-IR photons is less well understood is the behaviour of Tc agents at the
and can be applied to both pre-clinical in vivo and cellular sub-cellular level, since the image resolution of SPECT does
microscopy studies. However, it lacks the excellent tissue pene- not allow such determinations. Therefore intrinsically lumi-
9
9m
9
tration offered by nuclear imaging modalities.
nescent or fluorescently tagged
Tc SPECT agents are
The combination of radioimaging and fluorescence potentially helpful in correlating in vitro and in vivo imaging
1
0
imaging presents significant opportunities in the design of properties, and in determining their cellular uptake, traffick-
1
1
ing and localisation. From an inorganic chemistry perspec-
tive, non-radioactive rhenium is the obvious choice for devel-
oping structurally analogous and chemically related com-
a
School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10
AT, Cymru/Wales, UK. E-mail: popesj@cardiff.ac.uk, s.j.archibald@hull.ac.uk
1
2
3
b
plexes. Such an approach can be used to validate the develop-
Positron Emission Tomography Research Centre and Department of Biomedical
Sciences, University of Hull, Cottingham Road, Hull, HU6 7RX UK
13
99m
ment of appropriate chelates for
Tc and develop experi-
14
mental protocols for synthesis and purification. Indeed,
rhenium complexes are now attracting considerable attention;
in part for their potential as cancer therapy agents as well as
c
Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire, UK
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
1
5
c9dt04024b
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the existence of accessible radioisotopes appropriate for
1
6
radionuclide therapies/theranostic applications.
Binuclear
Re(I) carbonyl complexes have shown accumulation in mito-
1
7
chondria leading to cell death.
Re(I)/
Indeed
Tc(I) complex has also been described where the emis-
sive nature of the Re(I) fragment can be exploited in optical
a bimetallic
9
9m
1
8
studies. In the case of fluorescently tagged rhenium com-
plexes, these species can be assessed with respect to cellular
uptake using conventional confocal fluorescence microscopy
1
9
techniques.
Whilst substituted 1,8-naphthalimides have been previously
investigated in biological assays for their therapeutic appli-
2
0
Scheme 1 Design aspects of the compounds synthesised in this study
cations, more recent studies have shown that such species
can also be very useful fluorophores for fluorescence bio-
+
to provide a fluorophore functionalised chelate to the {M(CO)
3
}
unit (M
9
9m
1–6
=
Tc, Re). Six derivatives (L ) were synthesised with three different
2
1–23
imaging of cells.
The relative ease of functionalisation
linker units.
together with advantageous absorption and emission pro-
perties in the visible region have led to a large number of
reports on their application to cell imaging. For example,
recent studies have shown that the 1,8-naphthalimide struc-
ture can dictate intracellular localisation, including effective
2
4
mitochondrial targeting.
Since mitochondria have essential roles in energy pro-
duction, cell signalling, cell-cycle control and cell death, and
mitochondrial function can be disrupted in both ischaemic
heart cells and cancer cells, they are attractive targets. Thus
the development of a SPECT/fluorescence multimodal imaging
agents for targeting mitochondria would therefore allow for
in vitro, in vivo and ex vivo examination of myocardial function
2
5
and tumour growth. In this context, this current work
describes the development of 1,8-naphthalimide functiona-
9
9m
lised chelates suitable for labelling with
Tc(I) for mitochon-
drial targeted multimodal imaging. During the course of these
studies, work by Turnbull et al. has shown that a series of
related 4-amino-1,8-naphthalimide fluorophores can be com-
9
9m
plexed with Re(I)/ Tc(I). The overall charge of the complex
was shown to strongly influence the biological attributes of the
2
6
species.
Synthesis and characterisation of
ligands and complexes
Scheme 1 shows the different chelator structures that were syn-
thesised in this study. The compounds were designed to meet
the coordination chemistry requirements of M(I) (where M = Scheme 2 Synthesis of the precursors and ligands. Reagents and con-
ditions: (i) N-Boc-ethylenediamine, DMSO, heat; then TFA/DCM; (ii) 1,6-
Re, Tc) with a face-capping tridentate donor set, fulfilled by a
2
7
diaminohexane, DMSO, heat; (iii) 2-pyridinecarboxaldehyde, NaBH
OAc) , 1,2-dichloroethane; (iv) N-Boc(2,2’-(ethylenedioxy)diethylamine,
DMSO, heat; then TFA/DCM.
dipicolylamine (DPA) chelating moiety. This is linked to the
naphthalimide fluorophore for optical imaging via different
length spacers of variable hydro- and lipophilicity. Thus, a
series of six ligands were synthesised in three or four steps
(
3
from
4-chloro-1,8-naphthalic
anhydride
(Scheme
2).
Substitution at the 4-chloro position of the naphthalimide ring hyde in a one-pot reductive amination procedure gave the di-
1
–6
was achieved in DMSO at elevated temperature using an excess picolylamine derived target ligands, L , in each case. All
of diamine (often as the mono-Boc protected version). experimental details and spectroscopic characterisation data
Following cleavage of the Boc group (trifluoroacetic acid in di- for the synthesis of intermediates and ligands are included in
1
–6
chloromethane), treatment of N
with 2-pyridinecarboxalde- the Experimental section.
512 | Dalton Trans., 2020, 49, 511–523
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All isotopes of technetium are radioactive, and so natural Table 1 Solvent dependent intramolecular charge transfer absorption
2
8
and emission data for the ligands and rhenium(I) complexes
rhenium is commonly used as a chemical analogue to allow
the synthetic and purification protocols to be optimised prior
to the use of a radionuclide. The corresponding tricarbonyl
1
λ
nm
abs (ε/M− cm−1)/
em
λ /
a
b
c
Compound
nm
τ
obs/ns
ϕ/%
2
9
Re(I) complexes were isolated by reacting the precursor com-
pounds with fac-[Re(CO) (MeCN) ]BF to yield fac-[Re(CO) (L)]
BF as air stable solids. Characterisation of the complexes was
1
d
e
d
e
d
e
d
e
d
e
f
d
d
L
434 (9310)
28 (7800)
518
508
521
510
518
508
517
505
542
11.1
9.8
10.5
8.5
15
19
22
17
28
28
31
35
3
3
4
3
e
e
4
2
d
d
d
4
L
436 (10 420)
e
e
e
achieved using a range of spectroscopic and analytical tech-
niques. The expected facial geometry with respect to the three
carbonyl ligands was confirmed using IR spectroscopy. In all
427 (8160)
3
d
d
d
L
433 (7300)
9.7
e
e
f
e
4
35 (8240)
1.4, 8.8 (94%)
4
d
d
d
L
429 (8000)
9.5
8.1
e
f
e
e
cases two CuO stretches were observed between
430 (18 600)
5
f
040–1900 cm− which are attributed to the pseudo C3v sym-
1
L
448 (10 220)
3.4, 6.0 (64%)
39
2
d
d
d
d
4
29 (8910)f
521f
9.4f
12
6
f
metry of the complexes. In addition to this, the naphthalimide
L
448 (2620)
549
5.4
8
−
1
d
d
d
d
carbonyl stretches were observed at 1700–1625 cm and the
429 (7920)d
520
9.7
10.9
9.1
9.8
9.2
9.8
7.7
10.2
8.0
6.0
10.0
5.9
30
67
23
30
37
26
29
40
38
49
15
57
1
d
d
d
−
−1
[Re(CO)
Re(CO)
[Re(CO)
3
3
3
3
3
3
(L )]BF
4
4
4
4
4
4
430 (8560)
32 (7510)
515
[BF
4
]
counter anion at ca. 1055 cm . High resolution mass
e
e
e
e
4
503
2
d
e
d
d
d
spectrometry data were obtained for each complex identifying
the cationic fragment with the appropriate isotopic distri-
[
(L )]BF
433 (19 200)
430 (14 300)
430 (16 300)
519
e
e
e
504
3
d
e
d
d
d
1
(L )]BF
520
bution for rhenium. H NMR spectroscopy was able to clearly
e
e
e
4
34 (10 100)
512
4
d
e
d
d
d
establish the formation of the complex in each case. Firstly,
the methylene linkers of the dipicolylamine unit shift upon
coordination and become diastereotopic with an observed
[
[
[
Re(CO)
Re(CO)
Re(CO)
(L )]BF
431 (13 020)
425 (18 660)
517
e
e
e
483
5
f
f
f
f
(L )]BF
446 (7190)
29 (8100)
540
d
d
d
d
4
526f
2
8
6
f
f
f
coupling around 16 Hz. The aliphatic protons of the linker
unit are also mildly shifted in the complex versus the ligand.
The general pattern amongst the aromatic proton resonances
(L )]BF
445 (10 700)
448 (12 240)
539
d
d
d
d
516
11.1
17
a
b
Measurements obtained in aerated solutions; λex = 405 nm.
ex
λ =
c
is retained upon formation of the complex. Principally, data 295 nm. Using aerated MeCN solution of [Ru(bipy) ](PF ) as a refer-
3
6 2
d
e
f
1
3
ence. In acetonitrile. In chloroform. In water.
from C NMR studies allowed identification, in all cases, of
the rhenium-bound carbonyls (ca. 195 ppm) and the naphtha-
limide carbonyls (ca. 165 ppm).
Luminescence properties
Prior to radiolabelling and imaging studies, the electronic and
optical properties of these species were investigated (Table 1).
The UV-vis. spectra of all ligands were recorded in MeCN or
chloroform solutions. Because of the relative hydrophilicity of
5
6
the L /L systems, additional data in aqueous solution was
also obtained on these ligands and their complexes. The
absorption spectra showed some common features, with
higher energy absorptions associated with the naphthalimide
and pyridyl based π–π* transitions, and a lower energy, broad
peak around 440 nm which was assigned to an intramolecular
5
charge transfer (ICT) transition from the amine-functionalised Fig. 1 UV-vis. absorption spectrum for fac-[Re(CO) (L )]BF recorded in
3
4
naphthalimide chromophore. The position of the ICT peak water.
showed a slight shift that was dependent upon the amine sub-
stituent and was shown to be sensitive to the polarity of
solvent, as expected for a charge transfer transition. The 505–550 nm, giving Stokes’ shift >3500 cm−1, which is typical
absorption spectra of the corresponding Re(I) complexes (for of this class of fluorophore (for example, Fig. 2). Again, the
example, Fig. 1) were comparable, with only minor shifts of position of the peak was found to be moderately sensitive to
the ICT band upon formation of the cationic Re(I) complex the amine substituent (related to the relative electron donating
that are likely to be a result of the cationic charge perturbing power) and the solvent polarity. For example, measurements in
30
3
1
5
6
the ICT transition. Typically the molar absorption coeffi- aqueous media for L /L and their complexes resulted in a
cients (Table 1) of the ICT bands in the complexes were in the bathochromic shift of the emission maxima to ca. 540 nm,
−
1
−1
range of 7000–20 000 M cm
.
typical of positive solvatochromism (Fig. 2). Time-resolved
Steady state emission spectra were obtained using an experiments revealed lifetimes <15 ns in all cases, confirming
irradiation wavelength of 425 nm, corresponding to direct exci- the fluorescent nature of these ICT emitting species. Quantum
tation of the ICT band of the ligand. All ligands and complexes yields were obtained for each species in the various solvents,
5
6
showed an intense, featureless luminescence peak at and ranged 8–67%. For L /L and their rhenium(I) complexes
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5
Fig. 2 Luminescence spectrum for fac-[Re(CO)
3 4
(L )]BF recorded in
water (λex = 425 nm).
these values were relatively high (around 50%) for the aqueous
solutions. Taken together this gives a typical brightness value
−
1
−1
around 5000
M
cm . The naphthalimide conjugated
rhenium(I) complexes are thus well suited to cell imaging via
confocal fluorescence microscopy, with visible fluorescence,
good Stokes’ shifts and relatively high quantum yields giving
reasonably bright fluorophores.
Fig. 3 Preparation (top) and stability assessments (bottom) of radio-
5
5 +
3
labelled L : comparative HPLC traces for fac-[Re(CO) (L )] and fac-
99m
5 +
[
3
Tc(CO) (L )] .
Radiolabelling with technetium-99m
Table 2 Radiochemical
yields
for
the
formation
of
fac-
9
9m
5 +
Having established the Re(I) coordination chemistry and the
optical properties of the complexes, radiolabelling of selected
ligand systems was attempted with the SPECT imaging
[
Tc(CO)
3
(L )]
5
L conc. (mM)
30 min
60 min
90 min
(
gamma emitting) isotope technetium-99m. Radiolabelling 0.2
43
51
56
42
47
53
57
41
53
55
57
43
9
9m
+
0
.4
1.0
.0
studies were conducted using fac-[ Tc(CO)
was obtained from the reduction of [ TcO4] using the
method reported by Alberto et al.
olabel L (the ligand variant with an ethylene linker between
the naphthalimide and the dipicolylamine chelate unit) led to
the formation of a number of radioactive species indicative,
perhaps, of the decomposition of the ligand. Certainly, the
limited aqueous solubility of L was likely to be a hindrance to
efficient radiolabelling.
3
(H
2
O)
3
] , which
9
9m
−
2
3
2,33
Initial attempts to radi-
1
radiolabelling yield at 0.53–1.32 mM precursor concentrations.
Thus, optimised L radiolabelling conditions were fixed at
5
0
3
.53–1.32 mM precursor concentration, pH 7–8 (PBS), 70 °C,
0 min (Fig. 3).
1
Therefore, the focus of further radiochemistry experiments
was focussed upon the more hydrophilic naphthalimide L
9
9m
5 +
5
3
Stability and mitochondrial uptake of fac-[ Tc(CO) (L )]
(
the ligand variant incorporating the ethylenedioxodiamine Lipophilicity is an important parameter in dictating the cellu-
35
linker) which demonstrated good water solubility.
lar localisation of organometallic complexes. Lipophilicity
5
Preliminary experiments with water-soluble L indicated (log D7.4) measurements were carried out on the radiolabelled
9
9m
5 +
much better radiolabelling yields and limited degradation species fac-[ Tc(CO)
3
(L )] and showed a value of 0.67 ± 0.06.
Fig. 3), and therefore a range of conditions were tested to This suggests a species with suitable solubility for biological
optimise the radiolabelling reaction (Table 2). Varying quan- studies, but passive cell membrane permeability is likely to be
3
4
(
5
36
tities of lyophilised L (15 to 300 μg), were radiolabelled with less favourable. For drug-like species, such a value can also
freshly reduced fac-[ Tc(CO)
0
9
9m
+
37
3
(H
2
O)
3
]
(10–40 MBq, indicate that a renal clearance pathway will be observed. To
9
9m
5 +
.13–2.6 mM final precursor concentration, pH 7–8, buffered mimic in vivo conditions, formulated fac-[ Tc(CO) (L )] was
3
with 5 mM PBS) at 70 °C. The radiolabelling yield was deter- incubated with human serum at 37 °C. HPLC analysis did not
mined by integration of radio-HPLC traces and, at 30 min, reveal any significant instability up to 3 hours, indicating that
varied from 23% to 57%, plateauing at 55–57% for 30–150 μg the radiolabelled compound had sufficient stability to progress
(
0.26–1.32 mM) range while declining to 43% when the to preliminary in vivo imaging experiments.
5
99m
5 +
amount of L was increased to 300 μg (2.0 mM). Increasing the
3
To investigate fac-[ Tc(CO) (L )] behaviour towards mito-
reaction time to 60 min only had an effect on the lowest con- chondria, experiments were carried out using mitochondria
9
9m
5 +
centration (0.13 mM), with the yield increasing from 23% to freshly isolated from rat heart tissue. fac-[ Tc(CO)
3
(L )]
accumulated in the mitochondria to a higher extent (55.68 ±
up to 124–135 MBq) did not have any observable effect on the 6.5%) than complexes containing the cationic triphenylpho-
9
9m
+
3
3 2 3
9%. Similarly, a three-fold increase of fac-[ Tc(CO) (H O) ]
(
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sphonium mitochondria targeting group that has been fre-
3
8
quently used in mitochondrial targeting. This uptake was
highly dependent on mitochondrial membrane potential
(
0
MMP) as shown by a nearly complete abrogation (1.35 ±
.14%) when the MMP was depolarised using carbonyl cyanide
m-chlorophenylhydrazine (CCCP).
5
+
Cell imaging studies with fac-[Re(CO) (L )] and preliminary
3
9
9m
5 +
in vivo assessment of fac-[ Tc(CO) (L )]
3
Cellular cytotoxicity was assessed using U87 cells in vitro by
the MTT assay, giving a CC50 value of 72.7 μM (Fig. 4), which
indicates low cytotoxicity in vitro for the compound. Whole cell
5
+
mitochondrial uptake of fac-[Re(CO) (L )] was then studied
3
using confocal fluorescence microscopy in human breast
cancer cells (MCF-7) in culture. As a control, a benzyl chloride
based mitochondrial stain (MitoTracker deep red, MDR) was
used for dye co-localisation studies as its emission wavelength
5
+
(665 nm) is sufficiently different from fac-[Re(CO)
3
(L )]
(540 nm) thus allowing independent detection. Confocal fluo-
rescence microscopy studies into the mitochondrial localis-
5
+
3
ation for fac-[Re(CO) (L )] with MCF-7 cells gave an overlap
coefficient of 0.66 (Fig. 5) suggesting a good degree of co-
localisation.
Fig. 5 Confocal fluorescence microscopy images of MCF-7 cells incu-
bated with fac-[Re(CO) (L )] (top left) and MitoTracker deep red (top
3
right) and overlay (bottom; overlap coefficient = 0.66).
Having established the mitochondrial and cellular uptake
properties, a scoping in vivo experiment was carried out with
5
+
9
9m
5 +
3
fac-[ Tc(CO) (L )] in a naïve mouse using SPECT/CT
imaging (Fig. 6). Firstly, there were no signs of toxicity to the
mouse during the in vivo studies. The tracer showed rapid
clearance, predominantly via the biliary system along with a
moderate amount cleared renally. This is consistent with the
9
9m
5 +
log D of fac-[ Tc(CO) (L )] , as some renal clearance was
3
observed and more complex active transport mechanisms can
3
9
influence hepatobiliary clearance. Urine analysis demon-
strated that the tracer was of a relatively high stability (85.4 ±
1
.4%) and suitable for further in vivo analysis. From these
imaging studies, it was clear that cardiac uptake was not
9
9m
5 +
observed for fac-[ Tc(CO) (L )] . This suggests that in its
3
current form the tracer will not be appropriate for assessing
cardiac function in vivo and so structural modification would
be required to ensure uptake and retention.
Fig. 6 SPECT and CT overlaid images with the SPECT images acquired
9
9m
5 +
using the radiotracer fac-[
3
Tc(CO) (L )] . Sagittal and coronal views
show the localisation of the tracer after 22–60 min. Red arrow: gall
bladder; yellow arrow: biliary clearance into the biliary tract and small
intestine; green arrow: bladder; blue arrow: kidney.
5
+
3
Fig. 4 Cell viability assay (MTT) result for fac-[Re(CO) (L )] .
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rected and excitation spectra were instrument corrected. The
pulsed source was a nano-LED configured for 295 nm or
Conclusions
In conclusion, this study shows the design, synthesis and 372 nm output operating at 1 MHz. Luminescence lifetime
characterisation of a new class of radiotracer which is preferen- profiles were obtained using the JobinYvon–Horiba FluoroHub
tially taken up into the mitochondria of cells. The tracer com- single photon counting module and the data fits yielded the
bines a naphthalimide fluorophore with a technetium(I) tricar- lifetime values using the provided DAS6 deconvolution
bonyl unit to give a structure with excellent cellular and mito- software.
chondrial uptake characteristics. Cellular bioimaging shows
Standard radiolabelling protocol
good image quality in the MCF-7 cell line. The preliminary
9
9m
in vivo assessment of the aqueous soluble
Tc(I) compound All work carried out with ionising radiations was carried out
shows rapid clearance, aligning with the requirements for within the legal requirements of the Ionising Radiations
nuclear imaging to ensure background signal is minimised. Regulations 2017 and the Environmental Permitting
These results now justify further investigation of the tumour Regulations 2010. The studies followed local rules and regu-
uptake properties in vivo with appropriate tumour models and lations to ensure safe and compliant handling of open source
larger animal cohorts.
In its current form, fac-[ Tc(CO)
radioactive materials. The labelling precursor in a glass HPLC
(L )] does not demon- vial was dissolved in 50 μL of MeOH and was further diluted
9
9m
5 +
3
strate cardiac uptake in vivo. Therefore, our further studies will with 100 μL of 10 mM PBS solution. The solution was degassed
also consider structurally modified variants that can promote by sonication and purged with argon for 10 min. Up to ∼400
9
9m
+
3 2 3
and optimise this characteristic. During the course of these MBq of freshly reduced fac-[ Tc(CO) (H O) ] in a reduction
studies, Luyt and co-workers reported a peptide-functionalised medium (∼500 μL) was added, bringing the final precursor
naphthalimide-based fluorophore that can be used to target concentration to previously determined optimum 0.5–1.3 mM
4
0
CXCR4 expressing cells for potential dual mode imaging.
concentration range. The reaction mixture was incubated in a
This further suggests that the inherent fluorophore–chelate heating shaker at 70 °C mixing at 600 rpm for 30 min. After
design presented herein holds great promise for future biologi- which it was cooled down to room temperature and injected
cal and bioimaging applications.
onto semi-preparatory HPLC (ACE5 C18 5 Å 100 × 250).
Product containing fraction (RCY = 47 ± 18%, n = 3, corrected
for decay to the beginning of reaction) eluting at 12–14 min
was collected, diluted twice with deionised water and loaded
onto homemade SPE cartridge (Sep-Pak Light) containing
Experimental
General considerations
80–100 mg of Oasis C18 sorbent. The cartridge was washed
All reagents and solvents were commercially available and were with 2–3 mL of deionised water and dried by a current of inert
used without further purification if not stated otherwise. For gas. The product was eluted by 500 μL of ethanol, the solvent
1
13
the measurement of H and C NMR spectra a Bruker was evaporated under gentle heating aided by a current of
3
00
Fourier
(300 MHz), Bruker AVANCE HD III equipped with a inert gas and reformulated into PBS. For in vivo evaluation, the
BFFO SmartProbe™ (400 MHz) or Bruker AVANCE III HD with formulation was filtered using a 0.22 μm filter for sterility
BBO Prodigy CryoProbe (500 MHz) was used. The obtained prior to animal administration. The decay corrected prepara-
chemical shifts δ are reported in ppm and are referenced to tory yield was 21 ± 11% (n = 3) and the full process requires
the residual solvent signal. Spin–spin coupling constants J are less than 2 h from the start of synthesis.
given in Hz.
Lipophilicity
Low-resolution mass spectra were obtained by the staff at
Cardiff University. High-resolution mass spectra were carried Log P was measured using a standard shake-flask method.
out at the EPSRC National Mass Spectrometry Facility at Briefly, an aliquot of the tracer solution in ethanol (∼1 MBq)
Swansea University. High resolution mass spectral (HRMS) was dried in a 1.5 mL Eppendorf tube and dissolved in 1 mL of
data were obtained on a Waters MALDI-TOF mx at Cardiff 1 : 1 mixture of octanol/PBS. Solution was vigorously mixed in
University or on a Thermo Scientific LTQ Orbitrap XL by the vortex shaker for 5 min at room temperature and phases separ-
EPSRC UK National Mass Spectrometry Facility at Swansea ated by centrifugation at 14.5 krpm for 2 min. 50 μL of each
University. IR spectra were obtained from
a Shimadzu phase was diluted up to 1 mL and measured using an AGC.
IR-Affinity-1S FTIR. Reference to spectroscopic data are given
for known compounds. UV-Vis studies were performed on a
Serum stability
Shimadzu UV-1800 spectrophotometer as MeCN solutions (2.5 An aliquot of the tracer in ethanol (∼3 MBq) was dried, dis-
or 5 × 10− M). Photophysical data were obtained on a solved in 0.5 mL of human blood serum in a HPLC vial and
JobinYvon–Horiba Fluorolog spectrometer fitted with a JY TBX incubated while shaking at 600 rpm at 37 °C up to 3 hours.
picosecond photodetection module as MeCN solutions. Samples were taken at time 0, 30 min, 60 min, 1 h, 2 h and
Quantum yield measurements were obtained on aerated MeCN 3 h; the protein was precipitated by addition of double volume
5
3 6 2
solutions of the complexes using [Ru(bpy) ](PF ) in aerated of ice cold methanol and removed by centrifugation and the
MeCN as a standard (Φ = 0.016). Emission spectra were uncor- supernatant analysed by radio-HPLC.
516 | Dalton Trans., 2020, 49, 511–523
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Paper
5
In vivo SPECT imaging and stability
Flow cytometry. 1 × 10 cells per well were seeded in six-well
plates and cultured for 2–3 days until 70–80% confluent. Cell
media was removed and 1 mL 0.1% FCS-containing media was
added. Carbonyl cyanide m-chlorophenylhydrazone (CCCP,
Sigma Aldrich, UK) in DMSO (50 mM, 1 μL) was added into
three wells (1 mL total volume) and 0.1% DMSO was added to
the remaining three control wells (as a control). Plates were
placed inside a CO2 incubator for 30 min, followed by the
addition of compound (500 nM, 50 µL media) to both groups.
All animal procedures were approved by the University of Hull
Animal Welfare Ethical Review Body (AWERB) and carried out
in accordance with the Animals in Scientific Procedures Act 1986
and the UKCCCR Guideline 2010 by approved protocols follow-
ing institutional guidelines (Home Office Project License number
41
60/4549 held by Dr Cawthorne).
Female CD1 nude mice (25–30 g, Charles River, UK) were
induced with 5% and maintained under anaesthesia with 3%
−
1
2
Plates were incubated inside the CO incubator at 37 °C for
isoflurane (oxygen at 1 L min ). Temperature and respiration
were monitored throughout the scan. SPECT-CT acquisition
was carried out on a Mediso NanoSPECT/CTPLUS, equipped
with multi-pinhole pyramid collimators. Animals were injected
1
hour, subsequently, media was removed and the cells were
washed twice with ice cold PBS buffer. Cells were harvested
into PBS with cell scrapper and isolated by centrifugation at
200g for 5 min at 4 °C. The pellet was re-suspended in 300 μL
of PBS and the cell suspension transferred into polypropylene
FACS tubes (Falcon 2054) and analysed by a FACScan flow cyt-
ometer (BD Biosciences Europe, Erembodegem, Belgium). Dot
plot was used to gate cells and the FL-1 channel was used to
measure variation in fluorescence intensities. Mean fluo-
rescence intensities were used to quantify for comparison.
9
9m
5 +
3
i.v. with ≈30 MBq (200 µL) of fac-[ Tc(CO) (L )] 30 min
prior to a 25 min SPECT scan (energy window: 126.4–154.6
keV). To show anatomical co-registration, a CT scan (240 pro-
jections, 1 s exposure, 55 kVp X-rays) was acquired following
the SPECT scan. SPECT and CT images were reconstructed
with an iterative algorithm and with exact cone beam Filtered
Back Projection, respectively (HiSPECT, Scivis GmbH,
Germany).
After imaging experiments, mice urine was collected, pro-
teins were precipitated by addition of double volume of ice
cold methanol and removed by centrifugation at 14.5 krpm for
3
Confocal microscopy. MCF-7 cells (2.5 × 10 ) and H9c2 cells
3
(
4 × 10 ) were seeded in Mattak confocal dishes (P35G-1.2-20-
2
C, 35 mm ) and cultured for 2–3 days until 70–80% confluent.
After reaching desired cell density, the growth media was
replaced with 1 mL 0.1% FCS-containing media and cells were
5
8
min and supernatant was analysed by radio-HPLC indicating
5.4 ± 1.4% stability (n = 1, triplicate analyses).
5
+
treated with fac-[Re(CO) (L )] (500 nM, 50 µL) for 1 hour
3
inside a CO
MDR, 250 nM, 20 µL) was added 1 hour prior to compound to
obtain double labelled cells for mitochondrial co-localisation
2 2
incubator under 5% CO . Mitotracker deep red
(
In vitro analysis
Cell culture. MCF-7 cells were purchased from the European study. After 30 min, the cells were washed twice with 1 mL PBS
Collection of Cell Cultures and grown in RPMI-1640 media. and media containing 15 mM HEPES was added. Unstained
Media was supplemented with 10% fetal calf serum (FCS). control samples of cells were used to check for auto fluo-
Human glioblastoma cell line from (U87) from ATCC and it rescence. Live cell images were obtained using ZEN software
was cultured using DMDM media with 10% (v/v) heat inacti- on a Zeiss LSM 710 inverted confocal microscope, equipped
vated fetal bovine serum (FBS). Media and FCS were purchased with an incubator chamber at 37 °C. Images were obtained
from Gibco/Life Technologies, UK. Cells were maintained in using a Zeiss 63× water objective.
2
Nunc 75 cm tissue culture flasks (Fischer Scientific, UK)
To measure the degree of overlap between subcellular local-
5
+
inside a humidified 5% CO incubator at 37 °C. Both cell lines isation of fac-[Re(CO) (L )] and MDR, a colocalisation coeffi-
2
3
were divided bi-weekly for maintenance of stock at a divided cient was calculated following standard methods. Single-label
ratio of 1 : 8–1 : 10 for MCF-7 cells. For confocal and flow cyto- control samples were prepared to eliminate fluorescence overlap
metry experiments cells were counted and seeding densities between two fluorescence channels; in addition to fac-[Re
5
+
(below) were used.
3
(CO) (L )] or MDR, untreated cells were assessed in order to
Toxicity. Cells were seeded in 96 well plate with 1000 cells eliminate background fluorescence. Single-label control samples
per well in 200 µl growth medium. The plates were incubated were imaged under the same exposure settings as double-labelled
overnight at 37 °C allow cells to adhere. After 18 h media was samples. Exact coordinates were determined using crosshairs at
removed from the wells and 100 μl of treated media with X and Y coordinates from single-labelled controls and were iden-
various concentrations of compounds was added. The plates tical for double-labelled samples. Once the exact coordinates
were returned to the incubator. After 72 h, MTS reagent were determined, the ZEN software gave tables containing values
(
Promega, UK) 20 µl was added to the wells. Plates were incu- of co-localisation coefficient and overlap coefficient.
9
9m
5 +
bated for 3 h at 37 °C. Absorbance reading were taken at
Mitochondrial uptake of [ Tc(CO) (L )] . Cardiac mito-
3
4
90 nm using Synergy HT microplate reader (Biotek, USA). The chondria were isolated following a modified method based on
4
2
absorbance value of each triplicate were averaged and the that reported by Boehm et al. A male Sprague–Dawley rat was
media only absorbance was subtracted. Compounds reading anaesthetised with Dolethal and the heart was excised, washed
were normalised to the control reading. Empty plate and com- and trimmed in cold buffer. The trimmed heart was trans-
pound added background analysis was carried out to ensure ferred into a centrifuge tube containing 5 ml of ice cold buffer
low levels of non-specific fluorescence.
and then minced. The minced cardiac tissue suspension was
This journal is © The Royal Society of Chemistry 2020
Dalton Trans., 2020, 49, 511–523 | 517

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Paper
Dalton Transactions
+
transferred to
a
Teflon glass homogeniser (Scientific Hz, 2H), 4.01 (t, J = 4.8 Hz, 2H) ppm. LRMS ES found m/z =
+
Laboratory Supplies, UK) and a further 5 ml of ice cold buffer 275.03 [M ].
A was added (total 10 ml). 1 ml trypsin was added and gently
2
44
1
Synthesis of compound C . Prepared as for C except
homogenized using a tight fitting Teflon pestle (Scientific using 2-methoxyethylamine (0.90 g, 12.9 mmol), yielding C as
Laboratory Supplies, UK). After 15 min of trypsin digestion, a yellow solid (1.96 g, 79%). H NMR (400 MHz, CDCl ): δH
2
1
3
1
0 ml of trypsin inhibitor was added and mixed with three 8.68 (d, J = 7.0 Hz, 1H), 8.61 (d, J = 8.1 Hz, 1H), 8.52 (d, J = 8.2
passes of the tight fitting Teflon pestle. The tissue suspension Hz, 1H), 7.91–7.80 (m, 2H), 4.44 (t, J = 5.7 Hz, 2H), 3.73 (t, J =
+
was then centrifuged at 600g for 10 min at 4 °C. The super- 5.7 Hz, 2H), 3.38 (s, 3H) ppm. LRMS ES found m/z = 289.05
+
natant was transferred into another centrifuge tube and centri- [M ].
1
1
fuged again at 8000g for 10 min at 4 °C. The resultant pellet
Synthesis of N . C (0.3 g, 1.01 mmol) was dissolved in
was resuspended in 10 ml of isolation buffer B and again cen- DMSO (4 mL) and N-Boc-ethylenediamine was added (0.47 g,
trifuged at 8000g for 10 min at 4 °C. The mitochondrial pellet 2.93 mmol) and the solution heated at reflux for 16 hours
was then resuspended in 100 µl of respiratory buffer contain- under a nitrogen atmosphere. The solution was flooded with
ing 10 mM succinate. The total protein content of the isolated 30 mL of water and neutralised with 0.1 M HCl. The product
mitochondria was measured using Bio-Rad protein assay. 10 µl was then extracted with dichloromethane (3 × 15 mL) and
of the isolated mitochondria suspension was added into washed with water (3 × 10 mL) and brine (3 × 10 mL). The
3
90 µl of water (40 times dilution). A standard solution was solvent was then reduced to a minimum and precipitation
made with BSA to calibrate the protein amount. 60 µl of the induced by the dropwise addition of petroleum ether.
mitochondria suspension was added to 3 ml of Bio-Rad solu- Filtration of the precipitate followed by washing with diethyl
tion and 60 µl from each of the BSA stock solution dilutions ether (5 mL) yielded the Boc-protected intermediate product as
1
were also taken and added to 3 ml of Bio-Rad solution.
a bright orange solid (0.27 g, 65%). H NMR (300 MHz,
Mitochondrial protein concentration was shown to be CDCl
3
): δ
H
8.55 (d, J = 7.3 Hz, 1H), 8.42 (d, J = 8.5 Hz, 1H), 8.27
1
1
.23 µg µl− , as it was 40 times diluted, the original concen- (d, J = 8.3 Hz, 1H), 7.59 (t, J = 7.7 Hz, 1H), 7.19 (s, 1H), 6.55 (d,
−
1
tration was 49.46 µg µl . Hence, the total mitochondrial J = 8.3 Hz, 1H), 4.45 (t, J = 4.3 Hz, 2H), 3.97 (s, 2H), 3.65 (dd,
1
3
protein amount was 4946 µg in 100 µl. 150 µg protein was ali- J = 10.2, 5.3 Hz, 2H), 3.49–3.38 (m, 2H), 1.42 (s, 9H) ppm.
C
1
quoted into each Eppendorf. 100 µl of respiratory buffer con- { H} NMR (101 MHz, CDCl ): δ 163.89, 163.10, 150.08, 134.03,
3
C
taining 10 mM succinate added to give the control (n = 3 for 130.78, 129.32, 128.73, 124.48, 121.99, 120.37, 108.76, 103.95,
+
each tracer). 10 µM CCCP was added into 100 µl of respiratory 57.96, 41.47, 40.36, 37.31 ppm. LRMS ES found m/z = 400.19
+
buffer containing 10 mM succinate and was then added in [M + H] . Deprotection of the intermediate product was
remaining Eppendorf tubes. All the Eppendorf tubes were achieved by stirring the solution of the intermediate in 50%
transferred into the CO incubator containing 5% CO at 37 °C TFA in DCM for 24 hours under a nitrogen atmosphere. The
2 2
for 15 min and after that 0.5 MBq of tracer was added to the solvents were then removed under vacuum, and the residue
control and CCCP treated mitochondria. All of the Eppendorf dissolved in methanol and the solvent removed again. This
tubes were transferred back into the CO
and after that the assay was terminated by centrifugation at yellow solid (0.178 g, 60%). H NMR (300 MHz, d -DMSO): δ
2
incubator for 30 min was repeated in triplicate to yield the final product as an oily
1
6
H
8
000g for 5 min at 4 °C. The supernatant was collected and the 8.63 (d, J = 8.3 Hz, 2H), 8.42 (d, J = 7.2 Hz, 2H), 8.26 (d, J = 8.4
mitochondrial pellet was washed three times with ice cold res- Hz, 2H), 7.78 (s, 2H), 7.69 (t, J = 7.8 Hz, 2H), 6.84 (d, J = 8.5 Hz,
piratory buffer containing 10 mM succinate. After the final 2H), 4.84 (s, J = 46.7 Hz, 2H), 4.11 (t, J = 6.3 Hz, 4H), 3.73–3.51
1
3
1
wash, the mitochondrial pellet was resuspended into 1 ml of (m, 8H), 3.39 (s, 2H), 3.17 (s, 4H) ppm.
C{ H} NMR
PBS. The amount of radioactivity was measured using the (101 MHz, d -DMSO): δ 163.89, 163.10, 150.08, 134.03,
6
C
gamma counter (PerkinElmer Wallac wizard 3″). Percent 130.78, 129.32, 128.73, 124.48, 121.99, 120.37, 108.76, 103.95,
+
accumulation was quantified by using the ratio of activity in 57.96, 41.47, 40.36, 37.31 ppm. LRMS ES found m/z = 332.12
+
+
pellet to that in the pellet plus supernatant. The experiment [M + Na] . HRMS expected m/z = 322.1162 for [M + Na] found
was carried out three times using internal triplicates. The data m/z = 322.1163.
2
1
2
is presented as an average of each internal triplicate with the
standard deviation among triplicates.
Synthesis of N . Prepared as for N but using C (0.3 g,
1.0 mmol), yielding the intermediate product as a bright
orange solid (0.157 g, 38%). H NMR (400 MHz, CDCl ): δH
.51 (d, J = 7.3 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.4
1
3
Ligands and precursors
8
1
43
Synthesis of compound C . 4-Chloro-1,8-naphthalic anhy- Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.02 (s, 1H), 6.50 (d, J = 8.3 Hz,
dride (2 g, 8.6 mmol) was dissolved in ethanol (60 mL) and 1H), 5.03 (s, 1H), 4.36 (t, J = 5.6 Hz, 2H), 3.65 (t, J = 5.7 Hz,
ethanolamine (0.79 mL, 12.9 mmol) was added. The solution 2H), 3.58 (s, 2H), 3.39 (s, 2H), 3.32 (s, 3H), 1.41 (s, 8H) ppm.
1
3
1
was then heated at reflux for 12 h under a nitrogen atmo-
3 C
C{ H} NMR (101 MHz, CDCl ): δ 164.33, 158.65, 150.24,
sphere. Upon cooling to 0 °C precipitation occurred, and fil- 134.77, 131.32, 129.94, 127.14, 124.71, 122.77, 120.39, 103.30,
1
1
tration yielded C as a yellow solid (2.01 g, 85%). H NMR 80.85, 77.24, 69.84, 58.80, 46.87, 39.50, 38.96, 28.37 ppm.
+
(400 MHz, CDCl
3 H
): δ 8.70 (d, J = 6.8 Hz, 1H), 8.64 (d, J = 8.4 LRMS found m/z = 414.20 [M + H] . Deprotection yielded the
Hz, 1H), 8.54 (d, J = 7.6 Hz, 1H), 7.88 (m, 2H), 4.48 (t, J = 4.8 final product as an orange oil (0.103 g, 87%). H NMR
1
518 | Dalton Trans., 2020, 49, 511–523
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Dalton Transactions
400 MHz, CD OD): δ
Paper
(
3
H
8.41 (d, J = 7.6 Hz, 2H), 8.28 (d, J = 8.8 J = 4.8 Hz, 2H), 1.34 (s, 9H) ppm. LRMS found m/z = 389.18 [M
+
Hz, 1H), 7.65–7.49 (m, 2H), 6.78 (d, J = 8.7 Hz, 1H), 4.24 (t, J = + H] . Deprotection of the intermediate product was achieved
6
.0 Hz, 2H), 3.69 (t, J = 6.1 Hz, 2H), 3.59 (t, J = 6.0 Hz, 2H), 3.27 by stirring in 50% TFA in DCM (10 mL) for 24 hours under a
+
(m, 2H), 3.25 (s, 3H) ppm. LRMS found m/z = 336.13 [M + Na] . nitrogen atmosphere. The solvents were then removed under
+
HRMS expected m/z = 336.1319 for [M + Na] , found m/z = vacuum, and the residue dissolved in methanol and the
36.1320. solvent removed again. This was repeated in triplicate to yield
Synthesis of N . C (0.75 g, 2.73 mmol) was dissolved in N as a red oil (0.29 g, 91%). H NMR (300 MHz, CD
3
3
1
5
1
3
OD): δ
H
DMSO (4 mL) and 1,6 diaminohexane (0.4 mL, 4.1 mmol) was 8.31–8.19 (m, 2H), 8.09 (m, 1H), 7.43 (m, 1H), 6.61 (m, 1H),
added and the solution heated at reflux for 16 hours. The solu- 4.23 (m, 2H), 3.80 (t, J = 5.1 Hz, 2H), 3.72–3.64 (m, 6H),
tion was flooded with 30 mL of water and neutralised with 0.1 3.59–3.51 (m, 2H), 3.30–3.25 (m, 2H), 3.11–3.01 (m, 2H) ppm.
1
3
1
M HCl. The product was then extracted with dichloromethane
3 × 15 mL) and washed with water (3 × 10 mL) and brine (3 × 134.14, 133.04, 130.55, 128.31, 128.01, 127.62, 123.86, 108.00,
0 mL). The solvent was then reduced to a minimum and pre- 103.65, 70.03, 68.62, 66.54, 59.03, 42.69, 39.22 ppm. LRMS
C{ H} NMR (75 MHz, CD OD): δ 164.78, 150.78, 149.04,
3 C
(
1
+
cipitation induced by the dropwise addition of petroleum found m/z = 388.18 [M + H] . HRMS expected m/z = 388.1872
ether. Filtration of the precipitate followed by washing with for [M + H] , found m/z = 388.1857.
+
6
5
2
diethyl ether (5 mL) yielded the product as an orange solid
Synthesis of N . Prepared as for N but using C (0.75 g,
1
(
0.422 g, 44%). H NMR (300 MHz, CDCl ): δ 8.58 (d, J = 7.4 2.59 mmol), first yielding the intermediate product as a red oil
Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), (0.46 g, 35%). H NMR (400 MHz, MeOD) δ 8.35 (d, J = 7.8 Hz,
3
H
1
7
.69–7.57 (m, 1H), 6.75–6.66 (m, 1H), 5.36 (s, 1H), 4.51–4.40 1H), 8.19 (d, J = 8.6 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 6.70 (d, J =
(
2
(
1
4
=
3
m, 2H), 4.03–3.92 (m, 2H), 3.50–3.34 (m, 2H), 2.78–2.67 (m, 8.6 Hz, 1H), 4.23 (t, J = 6.1 Hz, 1H), 3.75 (t, J = 5.5 Hz, 1H),
1
3
1
H), 1.95–1.75 (m, 2H), 1.65–1.41 (m, 6H) ppm. C{ H} NMR 3.67–3.62 (m, 1H), 3.62–3.55 (m, 8H), 3.27 (s, 1H), 2.96 (t, J =
5
126 MHz, CDCl
3
): δ
C
165.58, 165.01, 150.88, 135.09, 131.38, 5.1 Hz, 1H), 1.36 (s, 9H) ppm. Deprotection yielded the N as a
1
30.03, 127.61, 124.36, 122.09, 120.31, 108.23, 104.03, 60.41, red oil (0.32 g, 88%). H NMR (400 MHz, CD OD): δ 8.35 (d, J
3
H
3.27, 42.02, 41.22, 32.52, 28.28, 26.80, 26.41. LRMS found m/z = 7.8 Hz, 2H), 8.19 (d, J = 8.6 Hz, 1H), 7.50 (app. t, J = 7.9 Hz,
+
356.20 [M + H] , HRMS expected m/z = 356.1896 found m/z = 1H), 6.70 (d, J = 8.6 Hz, 1H), 4.23 (t, J = 6.1 Hz, 2H), 3.75 (t, J =
56.1965.
Synthesis of N . Prepared as for N but using C (0.75 g, 2.96 (m, J = 5.1 Hz, 2H) ppm. C{ H} NMR (400 MHz,
.38 mmol) yielding the product as an orange solid (0.420 g, CD OD): δ 116.2, 165.6, 152.4, 135.7, 132.2, 131.0, 139.2,
2%). H NMR (400 MHz, CDCl ): δ 8.59 (d, J = 7.4 Hz, 1H), 125.5, 123.2, 121.7, 109.5, 105.2, 71.6, 71.4, 70.7, 70.1, 67.9,
5.5 Hz, 2H), 3.67–3.62 (m, 2H), 3.62–3.55 (m, 8H), 3.27 (s, 3H),
4
3
2
13
1
1
4
8
3
C
1
3
H
+
.47 (d, J = 8.3 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 7.7 58.9, 44.2, 40.5, 39.7 ppm. LRMS found m/z = 402.20 [M + H] .
+
Hz, 1H), 6.72 (d, J = 8.2 Hz, 1H), 5.32 (s, 1H), 4.45 (t, J = 5.8 Hz, HRMS expected m/z = 402.2023 for [M + H] , found m/z =
H), 3.75 (t, J = 5.8 Hz, 2H), 3.57–3.30 (m, 5H), 2.74 (t, J = 6.6 402.2017.
2
1
3
1
1
1
Hz, 2H), 1.91–1.75 (m, 2H), 1.61–1.39 (m, 6H) ppm. C{ H}
NMR (126 MHz, CDCl ): δ 165.04, 164.97, 164.41, 150.14, dichloroethane and 2-pyridinecarboxaldehyde (0.07 mL,
49.91, 134.82, 134.74, 131.30, 129.94, 129.87, 126.81, 126.50, 0.74 mmol) was added and the solution stirred for 2 hours
Synthesis of L . N (0.11 g, 0.37 mmol) was dissolved in 1,2-
3
C
1
1
1
2
24.56, 124.49, 122.62, 122.55, 120.21, 120.16, 109.40, 109.14, under a nitrogen atmosphere. Sodium trisacetoxyborohydride
04.15, 104.09, 69.71, 58.66, 43.33, 41.19, 38.87, 28.62, 28.47, (0.24 g, 1.11 mmol) was then added and the solution stirred at
+
6.90, 26.81, 26.40. LRMS found m/z = 370.21 [M + H] . HRMS room temperature for 18 hours. The solution was then neutral-
+
expected m/z = 370.2127 for [M + H] , found m/z = 370.2125.
Synthesis of N . C (0.75 g, 2.72 mmol) was dissolved in chloroform, washed with water (3 × 20 mL) and brine (3 ×
DMSO (5 mL) and N-Boc(2,2′-(ethylenedioxy)diethylamine 20 mL). The organic layer was collected then dried over MgSO4
3
ised with saturated NaHCO and the product extracted into
5
1
(
1.4 g, 5.64 mmol) was added and the solution heated at reflux and filtered, and the solvent removed to yield the product as
1
3
for 16 hours under a nitrogen atmosphere. Upon being an orange oil (0.13 g, 73%). H NMR (400 MHz, CDCl ):
allowed to cool the solution was flooded with 30 mL of water δH 8.80 (d, J = 8.9 Hz, 1H), 8.57 (d, J = 7.3 Hz, 1H), 8.52–8.49
and neutralised with 0.1 M HCl. The product was then (m, 2H), 8.35 (d, J = 8.6 Hz, 1H), 7.98 (s, 1H), 7.68–7.60
extracted with dichloromethane (3 × 15 mL) and washed with (m, 1H), 7.55–7.47 (m, 2H), 7.31 (d, J = 7.6 Hz, 2H), 7.12–7.07
water (3 × 10 mL) and brine (3 × 10 mL). The crude intermedi- (m, 2H), 6.48 (d, J = 8.1 Hz, 1H), 4.70 (s, 1H), 4.43–4.38 (m,
ate product was then purified further though the use of silica 2H), 3.95 (s, 4H), 3.93–3.89 (m, 2H), 3.36–3.31 (m, 2H),
1
3
1
gel column chromatography using 3 : 2 acetone hexane with 3.01–2.96 (m, 2H) ppm. C{ H} NMR (151 MHz, CDCl
% triethylamine as the eluent. The solvents were then δC 164.76, 164.11, 160.85, 150.53, 140.25, 134.54, 131.29,
removed under vacuum yielding the intermediate product as a 129.82, 127.09, 125.21, 125.00, 124.53, 122.60, 122.29, 120.43,
3
):
4
1
red oil (0.40 g, 30%). H NMR (300 MHz, CDCl
3
): δ
H
8.37 (d, J = 104.65, 70.46, 70.15, 69.79, 68.32, 67.72, 67.40, 58.72, 38.90,
.8 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.08 (d, J = 8.1 Hz, 1H), 3.67 ppm. LRMS found m/z = 482.22 [M + H] . HRMS expected
.46 (t, J = 7.5 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 6.03 (s, 1H), m/z = 482.2192 for [M + H] , found m/z = 482.2202. UV-vis.
.00 (s, 1H), 4.36 (t, J = 4.8 Hz, 2H), 3.92 (t, J = 4.5 Hz, 2H), (MeCN) λmax (ε/dm mol cm ): 434 (9300), 280 (19 600), 262
.82 (t, J = 5.1 Hz 2H), 3.64 (m, 4H), 3.57–3.45 (m, 4H), 3.28 (d, (20 400) nm.
+
6
7
5
3
+
3
−1
−1
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Dalton Trans., 2020, 49, 511–523 | 519

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Paper
Dalton Transactions
2
1
2
Synthesis of L . Prepared as for L , but using N (0.10 g, necarboxaldehyde (0.07 mL, 0.74 mmol) was added and the
0
.32 mmol), 2-pyridine carboxaldehyde (0.06 mL, 0.64 mmol), solution stirred for 2 hours under a nitrogen atmosphere.
and sodium trisacetoxyborohydride (0.203 g, 0.13 mmol). Sodium trisacetoxyborohydride (0.24 g, 1.11 mmol) was then
Recrystallization from chloroform yielded the product as a added and the solution stirred at room temperature for
1
yellow crystalline solid (0.08 g, 51%). H NMR (400 MHz, 18 hours. The solution was then neutralised with saturated
CDCl
3
): δ
.59–8.56 (m, 2H), 8.42 (d, J = 8.6 Hz, 1H), 7.91 (s, 1H), with water (3 × 20 mL) and brine (3 × 20 mL). The organic
.73–7.67 (m, 2H), 7.60–7.52 (m, 2H), 7.40–7.36 (m, 2H), layer was then dried over MgSO and filtered, and the solvent
H 3
8.83 (d, J = 8.2 Hz, 1H), 8.64 (d, J = 7.7 Hz, 1H), NaHCO and the product extracted into chloroform, washed
8
7
7
2
2
4
5
1
.18–7.13 (m, 2H), 6.55 (d, J = 8.7 Hz, 1H), 4.44 (t, J = 6.0 Hz, removed yielding L as an orange oil (0.13 g, 73%). H NMR
H), 4.22 (t, J = 5.9 Hz, 2H), 4.01 (s, 4H), 3.73 (t, J = 6.3 Hz, (400 MHz, CDCl ): δ 8.80 (d, J = 8.9 Hz, 1H), 8.57 (d, J = 7.3
H), 3.40 (s, 3H), 3.05 (t, J = 5.2 Hz, 2H) ppm. C{ H} NMR Hz, 1H), 8.52–8.49 (m, 2H), 8.35 (d, J = 8.6 Hz, 1H), 7.98 (s,
OD): δ 153.26, 150.32, 147.84, 134.27, 131.02, 1H), 7.68–7.60 (m, 1H), 7.55–7.47 (m, 2H), 7.31 (d, J = 7.6 Hz,
3
H
1
3
1
(101 MHz, CD
3
C
1
1
3
29.74, 128.47, 127.97, 124.60, 122.14, 120.86, 118.93, 109.67, 2H), 7.12–7.07 (m, 2H), 6.48 (d, J = 8.1 Hz, 1H), 4.70 (s, 1H),
03.90, 69.27, 57.54, 48.94, 40.24, 39.01, 38.77, 38.45, 37.74, 4.43–4.38 (m, 2H), 3.95 (s, 4H), 3.93–3.89 (m, 2H), 3.36–3.31
1
3
1
6.45, 22.65, 13.02, 10.02 ppm. LRMS found m/z = 512.23 for (m, 2H), 3.01–2.96 (m, 2H) ppm. C{ H} NMR (75 MHz,
+
+
[
3 C
M + O + H] . HRMS expected m/z = 512.2281 for [M + O + H] , CDCl ): δ 165.52, 165.05, 159.26, 150.55, 148.88, 136.61,
3
−1
found m/z = 512.2292. UV-vis. (MeCN) λ
cm ): 436 (10 420), 280 (19 800), 262 (20 500) nm.
(ε/dm mol
134.76, 131.23, 129.77, 127.74, 124.27, 123.24, 122.13, 120.49,
109.09, 103.98, 77.52, 77.09, 76.67, 70.29, 69.25, 68.64, 61.91,
max
−
1
3
1
3
Synthesis of L . Prepared as for L but using N (0.21 g, 60.60, 53.83, 43.35, 42.60 ppm. LRMS found m/z = 570.27 for
+
+
0
.565 mmol), 2-pyridine carboxaldehyde (0.13 mL, 1.12 mmol) [M + H] . HRMS expected m/z = 570.2711 for [M + H] , found
3
−1
−1
and sodium trisacetoxyborohydride (0.36 g, 1.68 mmol). m/z = 570.2708. UV-vis. (MeCN) λmax (ε/dm mol cm ): 429
Product was isolated as an orange oil (0.386 g, 95%). H NMR (8190), 279 (19 200, sh), 260 (21 000) nm.
1
6
5
6
(400 MHz, CDCl ): δH 8.48 (d, J = 4.7 Hz, 1H), 8.28 (d, J = 7.2
Synthesis of L . Prepared as for L but using N (0.06 g,
3
Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.59 0.15 mmol), 2-pyridine carboxaldehyde (0.03 g, 0.3 mmol) and
(
t, J = 7.6 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, sodium trisacetoxyborohydride (0.1 g, 0.45 mmol) and triethyl-
2
1
1
4
3
1
H), 7.11–7.06 (m, 1H), 6.42 (d, J = 8.5 Hz, 1H), 5.91 (s, 1H), amine (2 mL). L was isolated as a red oil (0.063 g, 72%). H
.36 (t, J = 4.1 Hz, 1H), 3.96 (t, J = 4.6 Hz, 1H), 3.76 (s, 2H), NMR (500 MHz, CDCl ): δ 8.47 (d, J = 7.3 Hz, 1H), 8.42 (d, J =
.23 (t, J = 5.6 Hz, 1H), 2.48 (t, J = 7.1 Hz, 1H), 1.70–1.64 (m, 4.3 Hz, 2H), 8.38 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 7.9 Hz, 1H),
3
H
1
3
1
H), 1.53–1.48 (m, 1H), 1.37–1.25 (m, 2H) ppm. C{ H} NMR 7.53 (t, J = 7.1 Hz, 2H), 7.45–7.38 (m, 3H), 7.06–7.02 (m, 2H),
): δ 164.25, 163.88, 158.88, 149.05, 147.87, 6.60 (d, J = 8.5 Hz, 1H), 6.24 (s, 1H), 4.35 (t, J = 6.0 Hz, 2H),
(
101 MHz, CDCl
3
C
1
1
6
2
47.50, 135.65, 135.43, 133.59, 130.08, 128.52, 125.53, 123.30, 3.87 (s, 4H), 3.79 (t, J = 4.7 Hz, 2H), 3.66 (t, J = 6.0 Hz, 2H), 3.61
21.96, 121.26, 121.22, 120.95, 119.47, 118.98, 107.92, 103.03, (d, J = 4.8 Hz, 4H), 3.55–3.52 (m, 2H), 3.48 (d, J = 4.8 Hz, 2H)
13
1
3.29, 60.72, 59.38, 53.17, 42.49, 41.50, 27.55, 25.90, 25.87, ppm. C{ H} NMR (126 MHz, CDCl
3
): δ
C
165.02, 164.38, 159.72,
+
5.66 ppm. LRMS found m\z = 554.28 for [M + O + H] . HRMS 150.04, 149.12, 136.60, 134.74, 131.39, 130.10, 127.08, 124.66,
+
expected m/z = 554.2753 for [M + O + H] , found m/z = 123.08, 122.95, 122.47, 122.15, 120.74, 110.21, 104.34, 70.48,
3
−1
−1
5
2
54.2762. UV-vis. (MeCN) λmax (ε/dm mol cm ): 433 (7300), 70.35, 69.95, 69.67, 68.71, 60.87, 58.92, 53.66, 43.28, 39.08. LRMS
+
79 (18 200, sh), 263 (20 400) nm.
found m/z = 584.28 [M + H] . HRMS expected m/z = 584.2867 for
4
1
4
+
Synthesis of L . Prepared as for L but using N (0.20 g, [M
+ H] , found m/z = 584.2873. UV-vis. (MeCN) λmax
3
−1
−1
0
.51 mmol), 2-pyridine carboxaldehyde (0.1 mL, 1.08 mmol) (ε/dm mol cm ): 429 (7920), 281 (19 600), 263 (20 600) nm.
1
1
and sodium trisacetoxyborohydride (0.34 g, 1.62 mmol).
Product isolated as an orange oil (0.194 g, 69%). H NMR 0.10 mmol) was dissolved in chloroform and fac-[Re
3 4
Synthesis of fac-[Re(CO) (L )]BF . Compound L (50 mg,
1
(400 MHz, CDCl
3 3 3 4
) δ 8.56–8.49 (m, 3H), 8.43 (d, J = 7.7 Hz, 1H), (CO) (MeCN) ]BF (54 mg, 0.11 mmol) was added and the
8
3
5
5
2
1
1
1
3
.07 (d, J = 8.6 Hz, 1H), 7.63 (t, J = 7.7 Hz, 2H), 7.59–7.49 (m, solution stirred at reflux under a nitrogen atmosphere for 18 h.
H), 7.16–7.09 (app. t, J = 6.4 Hz, 2H), 6.66 (d, J = 8.3 Hz, 1H), Upon being allowed to cool, the solvent was reduced to a
.37 (s, 1H), 4.42 (t, J = 5.8 Hz, 2H), 3.81 (s, 4H), 3.42–3.32 (m, minimum and the product precipitated upon the dropwise
H), 2.56 (t, J = 7.1 Hz, 2H), 1.96–1.67 (m, 4H), 1.66–1.52 (m, addition of hexane. The product was then filtered to yield fac-
1
3
1
1
1
H), 1.52–1.29 (m, 2H). C{ H} NMR (75 MHz, CDCl ): δ
[Re(CO) (L )]BF as an orange solid (21.5 mg, 35%). H NMR
3
C
3
4
64.83, 164.20, 159.79, 149.76, 148.91, 136.50, 134.58, 131.16, (300 MHz, CD
3
H
CN): δ 8.78 (d, J = 5.3 Hz, 2H), 8.54 (d, J = 7.5
29.76, 126.28, 124.46, 122.98, 122.71, 122.02, 120.11, 109.54, Hz, 1H), 8.44 (dd, J = 8.3, 5.4 Hz, 2H), 7.89 (t, J = 7.7 Hz, 2H),
04.13, 77.53, 77.10, 76.68, 69.85, 60.40, 58.76, 54.21, 43.54, 7.77–7.69 (m, 1H), 7.49–7.41 (m, 2H), 7.35–7.27 (m, 2fH), 6.95
8.91, 28.68, 26.92, 26.76 ppm. LRMS found m/z = 552.29 for (d, J = 8.5 Hz, 1H), 6.40 (s, 1H), 5.01–4.81 (m, 4H), 4.22 (dd, J =
+
+
[M + H] . HRMS expected m/z = 552.2955 for [M + H] , found 12.3, 6.4 Hz, 2H), 4.03–3.92 (m, 2H), 3.74 (t, J = 6.2 Hz, 2H)
3
−1
−1
13
1
m/z = 552.2969. UV-vis. (MeCN) λmax (ε/dm mol cm ): 429 ppm. C{ H} NMR (151 MHz, d -DMSO): δ 196.90, 195.60,
6
C
(
8000), 282 (14 200, sh), 263 (17 500) nm.
164.33, 163.59, 160.98, 152.34, 152.29, 150.52, 141.21, 141.20,
Synthesis of L . N (0.075 g, 0.19 mmol) was dissolved in 5% 141.15, 134.51, 131.30, 129.79, 129.02, 126.29, 125.18, 123.85,
triethylamine in 1,2-dichloroethane and (20 mL) and 2-pyridi- 122.64, 120.89, 109.47, 104.79, 67.77, 67.68, 67.42, 58.45,
5
5
520 | Dalton Trans., 2020, 49, 511–523
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Dalton Transactions
8.41, 41.93. LRMS found m/z = 752.15 for [M] . HRMS NMR (300 MHz, CDCl
Paper
+
5
3
): δ
H
8.54 (d, J = 5.0 Hz, 2H), 8.47 (d, J =
+
expected m/z = 752.1506 for [M] , found m/z = 752.1515. FT-IR 7.4 Hz, 1H), 8.41 (d, J = 8.4 Hz, 2H), 7.62–7.46 (m, 5H), 7.12 (t,
(
1
solid, selected cm− ): 2027 (CuO), 1904 (CuO), 1684 (CvO), J = 6.4 Hz, 2H), 6.73 (d, J = 7.6 Hz, H), 5.22 (d, J = 17.0 Hz, 2H),
1
−
3
−1
638 (CvO), 1057 (BF
cm ): 430 (8560), 281 (18 900, sh), (21 000) nm.
4
). UV-vis. (MeCN) λmax (ε/dm mol
4.48–4.37 (m, 4H), 4.01–3.90 (m, 8H), 3.84 (s, 4H), 3.66 (t, J =
5.1 Hz, 2H) ppm. C{ H} NMR (151 MHz, CDCl ): δ 195.75,
3 C
−
1
13
1
2
Synthesis of fac-[Re(CO)
3
(L )]BF
but using compound L (58 mg, 1.0 mmol). H 129.88, 127.58, 125.25, 124.96, 124.50, 122.30, 120.33, 109.29,
NMR (400 MHz, CDCl ): δH 8.68 (d, J = 8.0 Hz, 1H), 8.64 (d, J = 104.33, 77.23, 77.02, 76.81, 70.45, 70.13, 68.39, 67.71, 67.41,
4
. Prepared as for fac-[Re 195.36, 165.59, 165.12, 160.83, 150.51, 140.00, 134.96, 131.50,
1
2
1
(
3 4
CO) (L )]BF
3
+
5
7
5
5
3
1
1
7
3
.3 Hz, 2H), 8.59 (d, J = 7.3 Hz, 1H), 8.48 (d, J = 8.2 Hz, 1H), 62.49, 43.29, 42.71 ppm. LRMS found m/z = 840.20 [M + H] ,
+
.81–7.66 (m, 5H), 7.22–7.17 (m, 2H), 6.73 (d, J = 8.5 Hz, 1H), HRMS expected m/z = 840.2043 for [M + H] , found m/z =
.80 (d, J = 16.0 Hz, 2H), 4.52 (d, J = 16.0 Hz, 2H), 4.41 (t, J = 840.2029. FT-IR (solid, selected cm− ): 2031 (CuO), 1928
1
−
.8 Hz, 2H), 4.28–4.15 (m, 4H), 3.71 (t, J = 6.7 Hz, 2H), 3.37 (s, (CuO), 1683 (CvO), 1635 (CvO), 1058 (BF
4
). UV-vis. (MeCN)
1
3
1
λ
max (ε/dm mol cm−1): 429 (8100), 279 (13 300), 262 (18 000)
3
−1
H) ppm. C{ H} NMR (151 MHz, CDCl
3
): δ
C
195.74, 195.73,
64.76, 164.11, 160.85, 150.59, 150.53, 140.25, 134.54, 131.29, nm.
6
29.82, 127.09, 125.21, 125.00, 124.53, 122.60, 122.29, 120.43,
Synthesis of fac-[Re(CO)
3
(L )]BF
4
. Prepared as for fac-[Re
5
6
1
0.46, 70.46, 70.15, 69.79, 68.32, 67.72, 67.40, 58.72, 43.70, (CO)
3
(L )]BF
4
but using compound L (62 mg, 1.0 mmol). H
+
8.90 ppm. LRMS found m/z = 766.17 for [M] . HRMS expected NMR (300 MHz, CDCl ): δH 8.54 (d, J = 4.9 Hz, 2H), 8.50–8.34
3
+
m/z = 766.1657 for [M ], found m/z 766.1671. FT-IR (solid, (m, 3H), 7.62–7.46 (m, 5H), 7.11 (app. t, J = 5.7 Hz, 2H), 6.73
selected cm− ): 2029 (CuO), 1910 (CuO), 1684 (CvO), 1638 (d, J = 8.4 Hz, 1H), 5.23 (d, J = 16.6 Hz, 2H), 4.46 (s, 1H), 4.40
1
3
−1
−1
(
CvO), 1055 (BF4−). UV-vis. (MeCN) λmax (ε/dm mol cm ): (t, J = 6.3 Hz, 3H), 4.03–3.91 (m, 5H), 3.85 (s, 4H), 3.72 (t, J =
1
3
1
4
33 (9800), 280 (18 400, sh), 261 (22 500) nm.
6.1 Hz, 3H), 3.69–3.63 (m, 2H), 3.39 (s, 3H) ppm. C{ H} NMR
. Prepared as for fac-[Re (151 MHz, CD OD): δ 195.81, 194.75, 164.73, 164.19, 160.54,
CO) (L )]BF but using compound L (58 mg, 1.0 mmol). H 151.54, 151.12, 139.90, 134.49, 130.83, 129.85, 127.58, 125.12,
3
Synthesis of fac-[Re(CO)
3
(L )]BF
4
3
C
1
3
1
(
3
4
NMR (400 MHz, CDCl
3
): δ
H
8.68 (d, J = 8.0 Hz, 1H), 8.64 (d, J = 124.34, 122.95, 122.07, 120.19, 108.16, 103.83, 70.22, 70.10,
5
7
5
5
3
1
1
1
3
.3 Hz, 2H), 8.59 (d, J = 7.3 Hz, 1H), 8.48 (d, J = 8.2 Hz, 1H), 69.35, 69.27, 68.63, 68.04, 67.77, 57.48, 42.96, 38.36 ppm.
+
.81–7.66 (m, 5H), 7.22–7.17 (m, 2H), 6.73 (d, J = 8.5 Hz, 1H), LRMS found m/z = 854.21 [M] , HRMS expected m/z = 854.2196
+
−1
.80 (d, J = 16.0 Hz, 2H), 4.52 (d, J = 16.0 Hz, 2H), 4.41 (t, J = for [M] , found m/z = 854.2190. FT-IR (solid, selected cm ):
.8 Hz, 2H), 4.28–4.15 (m, 4H), 3.71 (t, J = 6.7 Hz, 2H), 3.37 (s, 2029 (CuO), 1908 (CuO), 1684 (CvO), 1645 (CvO), 1057
1
3
1
−
3
−1
−1
H) ppm. C{ H} NMR (151 MHz, CD OD): δ 197.13, 196.19, (BF4 ). UV-vis. (MeCN) λmax (ε/dm mol cm ): 430 (12 240),
3
C
66.18, 165.72, 161.91, 161.34, 153.23, 151.64, 141.75, 135.67, 281 (21 000), 261 (23 100) nm.
32.45, 131.10, 129.28, 127.20, 127.02, 126.08, 124.81, 124.73,
10.94, 105.56, 70.70, 68.94, 68.91, 58.94, 49.85, 40.47,
+
9.88 ppm. LRMS found m/z = 808.21 for [M] . HRMS expected Conflicts of interest
+
m/z = 808.2137 for [M] , found m/z = 808.2140. FT-IR (solid,
selected cm ): 2031 (CuO), 1944 (CuO), 1638 (CvO), 1072
−
1
There are no conflicts to declare.
−). UV-vis. (MeCN) λmax (ε/dm mol cm ): 434 (10 100),
3
−1
−1
(BF
4
2
79 (18 200), 256 (22 400) nm.
4
Acknowledgements
Synthesis of fac-[Re(CO) (L )]BF . Prepared as for fac-[Re
3
4
1
4
1
(
CO)
NMR (400 MHz, CDCl
Hz, 1H), 8.41 (d, J = 8.3 Hz, 1H), 7.71 (app. t, J = 7.9 Hz, 2H),
3
(L )]BF
4
but using compound L (62 mg, 1.0 mmol). H
AD, IAF and SJAP thank DSTL and Ministry of Defence for
financial support. The staff of the EPSRC Mass Spectrometry
National Service (Swansea University) are also thanked. We
also thank EPSRC for instrumentation grant EP/L027240/1. We
acknowledge the Daisy Appeal Charity (grant no. DAhul2011)
for funding, and thank Dr Assem Allam and his family for the
generous donation to help found the PET Research Centre at
the University of Hull.
3
): δ 8.66–8.54 (m, 3H), 8.52 (d, J = 7.2
H
7
1
4
=
1
1
1
5
.63 (app. t, J = 10.7 Hz, 3H), 7.15–7.10 (m, 3H), 5.73 (d, J =
6.0 Hz, 2H), 4.45 (d, J = 16.0 Hz, 2H), 4.34 (t, J = 6.3 Hz, 2H),
.21–4.08 (m, 4H), 3.64 (t, J = 6.4 Hz, 2H), 3.31 (s, 3H), 1.14 (t, J
1
3
1
3 C
7.1 Hz, 4H) ppm. C{ H} NMR (151 MHz, CD OD): δ
95.52, 195.30, 164.77, 164.25, 160.51, 159.92, 151.82, 150.21,
40.52, 140.34, 134.40, 130.92, 128.80, 127.73, 125.62, 125.43,
24.61, 123.40, 123.32, 109.53, 104.15, 69.29, 67.53, 67.50,
7.54, 48.47, 48.20, 48.10, 47.17, 46.56, 39.06, 38.48 ppm.
Notes and references
+
LRMS found m/z = 822.23 for [M] . HRMS expected m/z =
+
+
8
22.2301 for [M] , found m/z = 822.2290 for [M] . FT-IR (solid,
1 B. P. Burke, C. Cawthorne and S. J. Archibald, Philos.
Trans. R. Soc., A, 2017, 375, 0261.
2 S. Lutje, M. Rijpkema, W. Helfrich, W. J. G. Oyen and
O. C. Boerman, Mol. Imaging Biol., 2014, 16, 747–755.
3 (a) L. E. Jennings and N. J. Long, Chem. Commun., 2009, 28,
3511–3524; (b) S. Lee and X. Chen, Mol. Imaging, 2009, 8,
−
1
selected cm ): 2029 (CuO), 1907 (CuO), 1681 (CvO), 1639
CvO), 1055 (BF4−). UV-vis. (MeCN) λmax (ε/dm mol cm ):
3
−1
−1
(
4
31 (13 020), 276 (24 400, sh), 260 (26 100) nm.
5
3 4
Synthesis of fac-[Re(CO) (L )]BF . Prepared as for fac-[Re
1
5
1
(CO) (L )]BF but using compound L (62 mg, 0.10 mmol). H
3 4
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Dalton Trans., 2020, 49, 511–523 | 521

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Paper
Dalton Transactions
8
3
7–100; (c) S. R. Cherry, Annu. Rev. Biomed. Eng., 2006, 8, 18 A. François, C. Auzanneau, V. Le Morvan, C. Galaup,
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