A bisphosphonate for 19F-magnetic resonance imaging

Article abstract of DOI:10.1016/j.jfluchem.2016.02.008

¹⁹F-magnetic resonance imaging (MRI) is a promising technique that may allow us to measure the concentration of exogenous fluorinated imaging probes quantitatively in vivo. Here, we describe the synthesis and characterisation of a novel geminal bisphosphonate (¹⁹F-BP) that contains chemically-equivalent fluorine atoms that show a single and narrow ¹⁹F resonance and a bisphosphonate group that may be used for labelling inorganic materials based in calcium phosphates and metal oxides. The potential of ¹⁹F-BP to provide contrast was analysed in vitro and in vivo using ¹⁹F-MRI. In vitro studies demonstrated the potential of ¹⁹F-BP as an MRI contrast agent in the millimolar concentration range with signal-to-noise ratios (SNR) comparable to previously reported fluorinated probes. The preliminary in vivo MRI study reported here allowed us to visualise the biodistribution of ¹⁹F-BP, showing uptake in the liver and in the bladder/urinary system areas. However, bone uptake was not observed. In addition, ¹⁹F-BP showed undesirable toxicity effects in mice that prevent further studies with this compound at the required concentrations for MRI contrast. This study highlights the importance of developing ¹⁹F MRI probes with the highest signal intensity achievable. " 2016 The Authors.

Full text of DOI:10.1016/j.jfluchem.2016.02.008

Journal of Fluorine Chemistry 184 (2016) 58–64
Contents lists available at ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
19
A bisphosphonate for F-magnetic resonance imaging
Gavin D. Kennya, Karen P. Shawa, Saranja Sivachelvama, Andrew J.P. Whiteb,
Rene M. Botnara, Rafael T.M. de Rosalesa,
*
a
Division of Imaging Sciences & Biomedical Engineering, King’s College London, St Thomas’ Hospital, London SE1 7EH, UK
Department of Chemistry, Imperial College London, Exhibition Road, South Kensington, London SW7 2AZ, UK
b
A R T I C L E I N F O
A B S T R A C T
19F-magnetic resonance imaging (MRI) is a promising technique that may allow us to measure the
concentration of exogenous fluorinated imaging probes quantitatively in vivo. Here, we describe the
synthesis and characterisation of a novel geminal bisphosphonate (19F-BP) that contains chemically-
equivalent fluorine atoms that show a single and narrow 19F resonance and a bisphosphonate group that
may be used for labelling inorganic materials based in calcium phosphates and metal oxides. The
potential of 19F-BP to provide contrast was analysed in vitro and in vivo using 19F-MRI. In vitro studies
demonstrated the potential of 19F-BP as an MRI contrast agent in the millimolar concentration range with
signal-to-noise ratios (SNR) comparable to previously reported fluorinated probes. The preliminary in
vivo MRI study reported here allowed us to visualise the biodistribution of 19F-BP, showing uptake in the
liver and in the bladder/urinary system areas. However, bone uptake was not observed. In addition,19F-BP
showed undesirable toxicity effects in mice that prevent further studies with this compound at the
required concentrations for MRI contrast. This study highlights the importance of developing 19F MRI
probes with the highest signal intensity achievable.
Article history:
Received 22 November 2015
Received in revised form 20 February 2016
Accepted 24 February 2016
Available online 26 February 2016
Keywords:
19F-MRI
Bisphosphonates (BPs)
Magnetic resonance imaging (MRI)
Preclinical imaging
Fluorinated bisphosphonate
ã 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
protons. First, the lack of endogenous MR-visible fluorine provides
an unambiguous readout of the introduced fluorine-containing
MRI is a medical imaging technique that offers high-resolution
images of soft tissues without the need for ionising radiation. In
addition, and unlike other techniques such as those based on
radionuclides, it does not require the injection of contrast agents in
order to obtain meaningful images. However, for some imaging
procedures such as angiography or molecular imaging, chemical
compounds can be used to enhance the contrast of the specific
tissue of interest. In this context, one area that MRI currently lags
behind other imaging modalities, particularly positron emission
tomography (PET) and single photon emission computed tomog-
raphy (SPECT), is the quantitative measurement of the signal
provided by these contrast agents. This is a key requirement for
molecular imaging applications. Current contrast-based MR
techniques rely on the detection of imaging agents containing
paramagnetic ions such as gadolinium, manganese or iron.
However, interpretation of the results is difficult due to the
varying underlying signal hyper- and hypo-intensities in MRI. In
answer to this 19F-MRI has been implemented. The use of fluorine
as the nucleus for magnetic resonance has several advantages over
compounds location. In addition the 19F MR signal can be
quantified, giving a measure of the contrast agent’s concentration.
This is in contrast to paramagnetic contrast agents used in 1H-MRI
and based on Gd, Mn and particularly Fe, where in vivo absolute
quantification is not achievable.
The main uses of 19F-MRI in biomedical imaging to date has
been for cell tracking [1–5] visualisation of inflammation [6–9] and
for imaging angiogenesis [10,11] all using 19F nanoparticles. This is
an obvious choice due to the capacity of nanoparticles to carry the
many fluorine atoms required to obtain sufficient signal. More
recently attempts have been made to image smaller compounds by
modulating the 19F signal using lanthanide metals [12,13] and used
for the detection of gene expression [14]. Despite these early
promising results and clear advantages for molecular imaging
compared to 1H-MRI, 19F-MRI remains underused in clinical
practice. This is due to a major disadvantage, which is low
sensitivity [15]. As a consequence most 19F-MRI probes designed to
date need to have many fluorine atoms to provide enough signal in
the tissues of interest (ꢀ20–50 mM 19F). However, the number of
fluorine atoms that a molecule can carry is limited for several
reasons. First is solubility, as the fluorine content of a molecule
increases, the water solubility decreases. The second limitation is
the number of 19F signals, the ideal 19F-MRI contrast agent having
* Corresponding author.
E-mail address: rafael.torres@kcl.ac.uk (R. T.M. de Rosales).
http://dx.doi.org/10.1016/j.jfluchem.2016.02.008
0022-1139/ã 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

G.D. Kenny et al. / Journal of Fluorine Chemistry 184 (2016) 58–64
59
one single narrow resonance to maximise signal and avoid imaging
artifacts. To achieve this all the fluorine atoms must be in the same
chemical and magnetic environment. Another limitation of
19F-MRI is related to the long longitudinal relaxation times (T1)
of the fluorine nucleus (ꢀ1–2 s). This translates into long
acquisition times for the MRI procedure due to the 5–10 s required
between radiofrequency (RF) pulses, which results in long times or
more complex non-standard MRI sequences.
phosphite, triethylorthoformate and dibenzylamine were reacted
for 29 h at 150–160 ꢁC to yield the benzylated bisphosphonate (1).
The amino group of 1 was deprotected with H2 and 10% Pd/C
catalyst to yield 2. After removal of the catalyst, 2 was reacted with
2.9 equivalents of trifluoroacetic anhydride (TFAA) in dry DCM for
3 h. Excess TFAA was used in order to prevent low reaction yields
due to potential hydrolysis of the anhydride. After evaporation of
the volatiles and work-up, 3 was recrystallised from cold hexanes
in good yields (78%). The compound was characterised by NMR,
HR-MS and the structure confirmed by X-ray crystallography (Fig.1
and Fig. SI)
We are interested in developing 19F-MRI contrast agents for
molecular imaging that show single and narrow 19F resonances
and short T1 relaxation times. Previously we have shown that
1,1-bisphosphonates (BPs) bind very strongly to metabolically
active bone and calcium phosphate materials such as hydroxy-
apatite using SPECT and PET imaging [16–19]. In addition, we
found that BPs also bind very strongly to many nanomaterials
based on lanthanide metal oxides of the type M2O3 (M = Gd, Er,
among others) with known relaxation rate-enhancement prop-
erties [19]. We hypothesised that a fluorinated BP molecule
could be an useful tool in the development of 19F-MRI probes,
that would allow to combine of the amplification properties of
nanoparticle-based platforms (high numbers of equivalent
fluorine atoms) with the relaxation-enhancement properties of
lanthanide-based materials (short acquisition times) without
affecting their water solubility. In this way we could potentially
achieve 19F-MRI probes with high signal intensity and sensitivity
that could be imaged in a short time. In addition, their solution
and in vivo properties could be easily controlled by surface
modification using the same BP chemistry. In this work, we
report our first attempts at achieving this aim by synthesizing
and characterising a new fluorinated BP (19F-BP,Scheme 1) and
evaluate for the first time its properties as a single molecule for
19F-MRI in vitro and in vivo.
The ethyl-protected bisphosphonate group of 3 was depro-
tected by reacting with excess bromotrimethylsilane followed by
methanolisis at room temperature. The reaction gave quantitative
yields of 19F-BP as assessed by NMR and MS, confirming complete
removal of the ethyl protecting groups. 19F-NMR and 31P-NMR also
confirmed the stability of the trifluoromethyl and bisphosphonic
groups, respectively. The solubility properties of 3 changed from
hydrophobic to hydrophilic after deprotection, as expected for
bisphosphonic acids, and allowed us to perform our imaging
studies in water. One of the main advantages of this compound
over most 19F-MRI contrast agents reported to date based on
perfluorinated molecules is the chemical equivalence of its F
atoms. Non-equivalent F atoms result in broad and/or multiple
resonances that have a negative effect on the final 19F-MRI signal.
In 19F-BP, however, having
a
narrow single 19F resonance
(ꢂ76.15 ppm, v1/2 = 4.9 Hz), maximises imaging signal and mini-
mises the appearance of image artefacts.
2.2. In vitro MR imaging studies
Phantom MRI studies were performed to evaluate the contrast
properties of 19F-BP (Fig. 2). The compound was dissolved in water
at pH 7 at several concentrations (27, 54 and 108 mM) and imaged
in a preclinical 9.4 T MRI scanner. A clear concentration-dependent
increase in signal intensity and signal to noise ratio (SNR) was
found, demonstrating that 19F-BP can be imaged in the high mM
concentration range. Stability studies were also performed using
these samples. The 1H NMR and 19F-MRI spectra remained stable
for 5 h at pH 7 and 37 ꢁC, confirming the stability of 19F-BP at these
2. Results and discussion
2.1. Synthesis
The reaction scheme for the synthesis of 19F-BP is shown in
Scheme 1. Tetraethyl aminomethyl-bisphosphonate (2) was
synthesized following published methods [20,21]. Briefly, diethyl
Scheme 1. The synthetic scheme of 19F-BP. (i) 29 h at 150–160 ꢁC; (ii) H2, 10% Pd/C catalyst in EtOH, room temperature; (iii) 3 h in dry DCM; (iv) (a) 24 h, Me3SiBr (15 eq) in dry
DCM, room temperature (b) 1.5 h MeOH, 1.5 mL, room temperature.

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G.D. Kenny et al. / Journal of Fluorine Chemistry 184 (2016) 58–64
Fig. 1. The molecular structure of 3 (CCDC ID 960161).
conditions. This gave us confidence to study its biodistribution
properties in vivo.
Another potential reason for the lack of bone uptake observed
could be a low signal to noise ratio (SNR). SNR measurements are
important in 19F-MRI and provide a measure of sensitivity (i.e.
contrast achieved with amount of imaging agent injected). SNR
values of a phantom sample with 19F-BP were found to be in the
50–150 range (32 ꢃ 32 matrix size) and 15–40 range
(64 ꢃ 64 matrix size) for different slice thicknesses. The size of
the matrix size is indirectly proportional to the sensitivity, hence
the higher values obtained at 32 ꢃ 32. For the mouse studies these
values were found to be in the 10–40 and 2–12 range, and compare
favourably to other animal studies from Bible et al. [23] and
Giraudeau et al. [24] (Fig. 4). It is important to note, however, that
19F-BP was found to be toxic at concentrations required to achieve
in vivo MRI signal (97–119 mM). While other BPs used for nuclear
imaging such as 99mTc-MDP are required in micromolar concen-
trations to obtain image contrast, the amount of BPs required for
MRI contrast or therapy is much higher. Toxicity has been observed
in animal studies with an amino-bisphosphonate used for
therapeutic purposes and injected intravenously (alendronate),
at doses of 20 mg/kg. However, doses of 150 mg/kg are required for
detecting the 19F-MRI signal of 19F-BP (for a 20 g mouse). Hence,
toxicity is likely to be the result of the bisphosphonate and not the
trifluoromethyl group, although further studies are required to
confirm this. These results prompted us to abandon the study of
19F-BP for bone imaging and look for potential strategies in order
to increase its sensitivity.
2.3. In vivo MR imaging studies
Preliminary in vivo studies were carried out in a 9.4 T scanner
with a healthy mouse. We have recently shown that bifunctional
BPs accumulate in areas of high bone metabolism such as the end
of long bones and bone metastases using SPECT imaging [17,18].
Hence, we expected 19F-BP to accumulate in bone. However, after
intravenous injection, only signals in the bladder/urinary system
and liver areas were detected, the former most probably due to
renal excretion as expected for a molecule of this size (Fig. 3A)
although this cannot be confirmed with the data available. In
addition, uptake in other tissues/organs of the same area such as
the uterus cannot be ruled out. It is important to note that the 19
F
and 1H acquisitions were not performed simultaneously and each
modality was acquired with different slice thicknesses (19F is
5 times thicker than the 1H image), complicating the interpretation
of the images. Motion artifacts could also be responsible for the
suboptimal overlay of the two modalities. The signal observed in
the liver area (Fig. 3C), which is a much bigger organ and hence less
affected by these issues (Fig. 3C), is more conclusive to uptake by
this organ. Liver uptake is common for lipophilic molecules, and
since fluorination is known to increase the lipophilicity of
compounds, it is likely to be the result of the trifluoromethyl
group. We believe that the lack of bone uptake may be the result of
its high lipophilicity, compared to non-fluorinated BPs, resulting in
higher liver uptake, and/or fast renal clearance. Indeed, recent
reports support the notion that fluorinated groups increase the
renal excretion of molecules in vivo [22]. Another interesting
possibility is that bone binding could have resulted in a chemical
shift of the 19F resonance that could result in a lack of signal from
bone. However, the presence of the expected single resonance in
the broad sweep width spectrum performed prior to the imaging
session strongly suggests this is not the case.
2.4. Potential strategies to improve sensitivity
The most obvious strategy to improve the sensitivity of
19F-BP is to increase the number of F atoms in the molecule.
Interestingly, there are some recent synthetic strategies that
would allow us to synthesise
a similar BP with several
chemically-equivalent F atoms [22]. However, an increase in
fluorine content will likely have two main adverse effects. First
is solubility, as we anticipate the water solubility will decrease

G.D. Kenny et al. / Journal of Fluorine Chemistry 184 (2016) 58–64
61
Fig. 2. In vitro MR imaging study. Top: 1H and 19F MRI phantom study with vials of 19F-BP at increasing concentrations (27, 54 and 108 mM) in water from left to right, with
water above. Bottom: graph showing the increase of signal to noise ratio (SNR) with increasing probe concentration. The fit is a smooth curve fit intended to represent the
trend. Error bars are the result of 3 ROI image analyses.
and eventually may result in water-insoluble compounds. The
second effect is related to this lower hydrophilicity. We have
observed a high degree of liver uptake and hence lipophilicity
with a trifluoromethyl group, addition of more fluorine atoms
will probably worsen this effect. Another potential adverse effect
would be the observed increased in vivo rate of excretion of
fluorinated agents others and we have observed [22]. A recent
proposed method to improve the sensitivity of 19F-MRI contrast
agents is by positioning the F atoms near a lanthanide in order
to enhance their relaxation rates. This technique has been
recently explored by Parker and Blamire et al. showing this
strategy can result in lower acquisition times and detection
limits by as much as 2 orders of magnitude [12,13,25]. We
hypothesised that, given the known ability of BPs to chelate Ln3+
metals and lanthanide oxide materials [19], we could explore
this property to enhance the relaxation rate of 19F-BP and hence
increase its sensitivity. This method, of course, would not be
useful for bone imaging, unless other bifunctional BPs that
contain a Ln3+ binding group such as a macrocycle chelate
(leaving the BP free to bind to bone mineral or inorganic
material) between the F-containing motif and the BP are
designed. However, it could provide a very useful method to
label lanthanide-containing nanomaterials with large numbers
of 19F atoms and fast acquisition times for other purposes such
as cell tracking or molecular imaging using 19F-MRI.
A
preliminary in vitro MR study in which we measured the
longitudinal and transverse relaxation rates (R1 and R2) of 19F-BP
in the absence and presence of 1 molar equivalent of different
lanthanide salts (Dy3+, Er3+, Gd3+, Ho3+ and Tb3+) supports the
potential of this approach as the presence of Ln3+ metals in the
solution enhance both relaxation rates by as much as 3 orders of
magnitude (supporting information). It is important to note,
however, that well-defined and characterised 19F-BP-Ln3+ com-
plexes would be required in order to validate these findings. We
believe this is a strategy that would be particularly useful in
conjunction with nanoparticle systems that can combine large
numbers of ꢂꢂCF3 groups with lanthanide metals at the surface
and the required distance from each other. Using this combina-
tion, high sensitivity (signal/mole contrast agent) may be
achieved thanks to the high numbers of chemically-equivalent
19F atoms (hundreds to thousands for a spherical nanometer size
particle) with the relaxation capabilities of paramagnetic metals.
In addition to the use of paramagnetic ion relaxation, the
sensitivity could be further increased in the future by using

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G.D. Kenny et al. / Journal of Fluorine Chemistry 184 (2016) 58–64
Fig. 3. Animal MR imaging study (see Section 4 for details). A mouse was injected with 19F-BP i.v. (108 mM in PBS buffer) and imaged using 1H (left column) and 19F MRI
(middle column), which were overlayed to determine location (right column). Top row is an axial slice through the bladder/urinary tract area, second row an axial slice
through the knees (arrows) and the third row an axial slice through the liver. A vial containing a known amount of 19F-BP was positioned next to the animal for reference.
more efficient MR protocols such as ultrafast sequences recently
developed [26].
was found to be toxic at the concentrations used in this study. From
these results it is clear that, while 19F-BP may not be useful for
bone imaging by itself it may be an useful compound to provide 19
F
3. Conclusions
signal to many inorganic materials of known affinity towards BPs
such as calcium phosphates (i.e. hydroxyapatite) and metal oxides,
as our recent work suggests. Future work is aimed at using 19F-BP
and related BPs to fully exploit this approach.
19F-BP was successfully synthesised and characterised. The
compound is water soluble and stable and shows a single and
narrow fluorine resonance ideally suited for 19F-MRI. Phantom
studies show that 19F-BP can be imaged using a 9.4 T magnet in the
high mM range with SNR ratios similar to other reported probes.
4. Experimental
An in vivo 19F-MRI study strongly suggests that F-BP was rapidly
4.1. Materials
19
excreted renally although uptake by other organs/tissue in the area
cannot be completely ruled out with our data. Uptake in the liver
was also observed which is probably a result of the lipophilicity of
the trifluoromethyl group. This data suggests that the lack of bone
uptake observed, the natural target of BPs, may be due to the
presence of the fluorinated group resulting in fast clearance, as
other studies have recently found [22]. More importantly, 19F-BP
Reagents and starting materials were obtained from commer-
cial sources and used as received unless otherwise noted. Organic
solvents were of HPLC grade. Water (Type I, 18.2 M
Vcm) was
obtained from an ELGA Purelab Option-Q system. Dittmer-Lester’s
TLC reagent for the detection of phosphorus was prepared
following the original literature protocol [27]. NMR spectra were

G.D. Kenny et al. / Journal of Fluorine Chemistry 184 (2016) 58–64
63
Fig. 4. Phantom and animal MR imaging study SNR values. SNR values were calculated for a variety of imaging parameters: matrix size (32 ꢃ 32, top; 64 ꢃ 64, bottom) and
slice thickness (x-axis), using 19F MRI in the phantom, bladder and liver. Error bars are the result of 3 ROI image analyses.
obtained in a 400 MHz Bruker Avance III (Germany). 1H chemical
shifts are referenced with respect to the residual solvent peak (dH
4.79 ppm, D2O; 7.26 ppm CDCl3) [28]. 31P resonances were
((P(OEt)3)2ꢂꢂCHꢂꢂNHꢂꢂ)), 16.0 (bs, (ꢂꢂP(O)(OCH2CH3)2)); 31P{1H}-
NMR (161.9 MHz, CDCl3, 298 K) dP (ppm) 14.02; 19F-NMR (376 MHz,
CDCl3, 298 K) dF (ppm) ꢂ76.47; HR-MS (ESI) 400.0939 (M + H+,
found), 400.0932 (M + H+, calculated). 422.0759 (M + Na+, found),
422.0721 (M + Na+, calculated).
referenced to an external solution of 85% H3PO4 (
dP 0 ppm). 13C
chemical shifts were referenced to the residual solvent peak (dC
77.16 ppm, CDCl3) or left unreferenced (D2O). 19F resonances were
referenced to an external solution of TFA (dF ꢂ78.5 ppm). High-
resolution mass spectra (HR-MS) were obtained using an Agilent
6500 Accurate-Mass Q-TOF LC–MS system using electrospray
ionization. Tetraethyl((dibenzylamino)methylene)bisphosphonate
(1) and tetraethyl(aminomethylene)bisphosphonate (2) were
synthesised following published methods [20,21].
4.2.2. ((2,2,2-trifluoroacetamido)methylene)bisphosphonic acid.
19F-BP
3 (89 mg, 0.22 mmol) was dissolved in dry drychloromethane
(200 cm3) under nitrogen and the flask cooled to 0 ꢁC. After 5 min,
bromotrimethylsilane (0.444 cm3, 3.3 mmol) was added dropwise
over 5 min. The ice bath was then removed and the solution was
left stirring under nitrogen at room temperature for 24 h during
which time the solution turned yellow. The volatiles were then
removed under reduced pressure and the residue dissolved in
1.5 cm3 of methanol, resulting in a colourless solution. The reaction
was left stirring at room temperature for a further 1.5 h followed by
evaporation under reduced pressure yielding the product in
quantitative yield as a clear sticky oil.
4.2. Syntheses
4.2.1. Tetraethyl((2,2,2-trifluoroacetamido)methylene)
bisphosphonate. 3
2 (200 mg, 0.66 mmol) was dissolved in dry drychloromethane
(200 cm3) under nitrogen and the flask cooled to 0 ꢁC. After 5 min,
trifluoroacetic anhydride (0.274 cm3, 1.9 mmol) was added in small
portions over 2 min. The ice bath was then removed and the
solution was left stirring at room temperature for 2 h during which
time the reaction mixture turned slightly yellow. The volatiles
were then removed under reduced pressure leaving a clear yellow
residue. This residue was dissolved in 2 cm3 of dichloromethane
and to this mixture were added increasing amounts of a 1%
solution of sodium bicarbonate followed by shaking, until the pH of
the aqueous layer was 7 (ꢀ10 cm3). The organic layer was
separated and washed with 3 cm3 of water, dried over sodium
sulfate, filtered and evaporated under reduced pressure. The
residue recrystallised from hexanes after 24 h standing at 4 ꢁC,
yielding large quantities of X-ray diffraction-quality crystals (78%
yield).
1H NMR (D2O, 400.3 MHz, 298 K) dH (ppm) 4.60 (t, 2JH-P = 22 Hz,
1H, ((P(O)(OH)2)2ꢂꢂCHꢂꢂNHꢂꢂ)); 13C NMR (D2O, 100.7 MHz, 298 K)
2
dC (ppm) 158.3 (q, JC-F = 40 Hz, (ꢂꢂNHꢂꢂCOꢂꢂCF3)), 115.8 (q, JC-
F = 285 Hz, (ꢂꢂNHꢂꢂCOꢂꢂCF3)), 47.3 (t, JC-P = 136 Hz, ((P
(OH)2)2ꢂꢂCHꢂꢂNHꢂꢂ)); 31P{1H}-NMR (161.9 MHz, D2O, 298 K) dP
(ppm) 12.07; 19F-NMR (376 MHz, D2O, 298 K) dF (ppm) ꢂ76.15; HR-
MS (ESI) 287.9664 (M + H+, found), 287.9650 (M + H+, calculated).
4.3. X-ray crystallography
Crystal data for 3: C11H22F3NO7P2, M = 399.24, triclinic, P-1
(no. 2), a = 10.2420(5), b = 10.2485(5), c = 10.5343(5) Å,
(4), = 71.735(4),
= 70.761(4)ꢁ, V = 930.40(9) Å3, Z = 2, Dc = 1.425
g cmꢂ3 ) = 2.700 mmꢂ1, T = 173 K, colourless blocks, Ox-
(Cu-K
a= 65.615
b
g
,
m
a
1H NMR (CDCl3, 400.3 MHz, 298 K) dH (ppm) 4.16 (m, 8H,
ford Diffraction Xcalibur PX Ultra diffractometer; 3670 indepen-
dent measured reflections (Rint = 0.0228), F2 refinement,
29R1(obs) = 0.0352, wR2(all) = 0.0972, 3257 independent observed
(ꢂꢂP(O)(OCH2CH3)2)),
3.56
(t,
2JH-P = 22 Hz, 1H,
((P(O)
(OEt)2)2ꢂꢂCHꢂꢂNHꢂꢂ)), 1.34 (s, 12H, (P(O)(OCH2CH3)2)); 13C NMR
2
(CDCl3, 100.7 MHz, 298 K) dC (ppm) 159.1 (q, JC-F = 40 Hz,
absorption-corrected reflections [|Fo| > 4
s
(|Fo|),
2
umax = 145ꢁ],
(ꢂꢂNHꢂꢂCOꢂꢂCF3)), 115.6 (q, JC-F = 291 Hz, (ꢂꢂNHꢂꢂCOꢂꢂCF3)),
249 parameters. Crystallographic data for the structures in this
paper have been deposited with the Cambridge Crystallographic
2
65.3 (t, JC-P = 3 Hz, (ꢂꢂP(O)(OCH2CH3)2)), 43.9 (t, JC-P = 148 Hz,

64
G.D. Kenny et al. / Journal of Fluorine Chemistry 184 (2016) 58–64
Data Centre (CCDC ID 960161). Copies of the data can be obtained,
free of charge, on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK (Fax: +44 1223 336033 or e-mail: deposit@ccdc.cam.
ac.uk).
funded by the CRUK and EPSRC in association with the MRC and
DoH (England). GDK was also part funded by the Rosetrees Trust.
The authors acknowledge financial support from the Department
of Health via the National Institute for Health Research (NIHR)
comprehensive Biomedical Research Centre award to Guy’s & St
Thomas’ NHS Foundation Trust in partnership with King’s College
London and King’s College Hospital NHS Foundation Trust.
4.4. Relaxation rate measurements
The relaxation times T1 and T2 of 19F in 19F-BP and 19F-BP + Ln3+
mixtures were measured in H2O at pH 7 at 400 MHz on a Bruker
Avance (Bruker, Ettlingen, Germany) and converted to the R1 (1/T1)
and R2 (1/T2) rates. T1 measurements were performed using an
inversion recovery technique with 8 inversion times between
0.001 and 4 s, TR = 7 s and 256 averages. T2 measurements were
performed with a spin echo technique with 12 TEs between 0.002
and 0.2 s, TR = 7 s and 8 averages. Analysis was performed using Top
Spin software (Bruker, Ettlingen, Germany).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.
jfluchem.2016.02.008.
X-ray crystallography data and relaxation rate measurements of
19F-BP in the presence and absence of lanthanide metal salts
(Table S1).
4.5. Magnetic resonance imaging (MRI)
References
4.5.1. Phantom imaging
[1] K.C. Partlow, J. Chen, J.A. Brant, A.M. Neubauer, T.E. Meyerrose, M.H. Creer, J.A.
Nolta, S.D. Caruthers, G.M. Lanza, S.A. Wickline, FASEB J. 21 (2007) 1647–1654.
[2] P. Boehm-Sturm, L. Mengler, S. Wecker, M. Hoehn, T. Kallur, PLoS One 6 (2011)
e29040.
19F-BP at different concentrations (27, 54 and 108 mM) in
250 mL PCR tubes were positioned in a 9.4T Bruker Avance vertical
bore scanner using a quadrature volume coil (Bruker, Ettlingen,
Germany) alongside a PCR tube containing water. For 1H imaging
for localisation a RARE sequence was used with TR = 1500 ms,
TE = 8.5 ms, NSA = 1, matrix = 256 ꢃ 256, FOV = 30 ꢃ 30 mm, slc = 1
mm. For the 19F imaging the coil was tuned to the 19F resonance
frequency and a spin echo sequence used with a TR = 3000 ms,
TE = 7.6 ms, NSA = 100, matrix = 32 ꢃ 32, FOV = 30 ꢃ 30 mm, slc = 6
mm, total scan time = 2 h 40 min.
[3] F. Bonetto, M. Srinivas, A. Heerschap, R. Mailliard, E.T. Ahrens, C.G. Figdor, I.J.M.
de Vries, Int. J. Cancer 129 (2011) 365–373.
[4] M. Srinivas, L.J. Cruz, F. Bonetto, A. Heerschap, C.G. Figdor, I.J.M. de Vries,
Biomaterials 31 (2010) 7070–7077.
[5] M. Srinivas, A. Heerschap, E.T. Ahrens, C.G. Figdor, I.J.M. de Vries, Trends
Biotechnol. 28 (2010) 363–370.
[6] U. Flögel, Z. Ding, H. Hardung, S. Jander, G. Reichmann, C. Jacoby, R. Schubert, J.
Schrader, Circulation 118 (2008) 140–148.
[7] U. Flögel, S. Su, I. Kreideweiß, Z. Ding, L. Galbarz, J. Fu, C. Jacoby, O. Witzke, J.
Schrader, Am. J. Transplant. 11 (2011) 235–244.
[8] R. Southworth, M. Kaneda, J. Chen, L. Zhang, H. Zhang, X. Yang, R. Razavi, G.
Lanza, S.A. Wickline, Nanomed. Nanotechnol. Biol. Med. 5 (2009) 359–367.
[9] Y.T. Lim, M.Y. Cho, J.-H. Kang, Y.-W. Noh, J.-H. Cho, K.S. Hong, J.W. Chung, B.H.
Chung, Biomaterials 31 (2010) 4964–4971.
4.5.2. Animal imaging
All animal experiments were performed with licences issued in
accordance with the United Kingdom Animals (Scientific Proce-
dures) Act 1986 (UK). One female Balb/c mice (Charles River,
Edinburgh, UK), 8–10 weeks old, was anaesthetised using 5% and
maintained with 1–2% isoflurane, and injected with 100 mL
compound (108 mM in PBS) via the tail vein before being
[10] E. Waters, J. Chen, J. Allen, H. Zhang, G. Lanza, S. Wickline, J. Cardiov, Magn.
Reson. 10 (2008) 43.
[11] C. Giraudeau, F. Geffroy, S. Mériaux, F. Boumezbeur, P. Robert, M. Port, C. Robic,
D. Bihan, F. Lethimonnier, J. Valette, Angiogenesis 16 (2013) 171–179.
[12] K.H. Chalmers, M. Botta, D. Parker, Dalton Trans. 40 (2011) 904–913.
[13] K.H. Chalmers, E. De Luca, N.H.M. Hogg, A.M. Kenwright, I. Kuprov, D. Parker, M.
Botta, J.I. Wilson, A.M. Blamire, Chem. Eur. J. 16 (2010) 134–148.
[14] S. Mizukami, H. Matsushita, R. Takikawa, F. Sugihara, M. Shirakawa, K. Kikuchi,
Chem. Sci. 2 (2011) 1151–1155.
transferred to the MRI scanner (9.4T Bruker Avance vertical bore
scanner using
a quadrature volume coil (Bruker, Ettlingen,
Germany)). For 1H imaging a FLASH sequence was used with
TR = 350 ms, TE = 5.4 ms, FA = 40ꢁ, NSA = 5, matrix = 256 ꢃ 256,
FOV = 30 ꢃ 30 mm, slc = 1 mm, 30 slices. For the 19F imaging the
coil was tuned to the 19F resonance frequency and a RARE sequence
used with a TR = 1500 ms, TE = 8.5 ms, RARE factor = 4, NSA = 200,
matrix = 32 ꢃ 32, FOV = 30 ꢃ 30 mm, slc = 5 mm, 6 slices, total scan
time = 30 min. In addition the same sequence was run, but with
FOV = 64 ꢃ 64, which had a total scan time of an hour.
[15] E. Terreno, D.D. Castelli, A. Viale, S. Aime, Chem. Rev. 110 (2010) 3019–3042.
[16] L. Sandiford, A. Phinikaridou, A. Protti, L.K. Meszaros, X. Cui, Y. Yan, G.
Frodsham, P.A. Williamson, N. Gaddum, R.M. Botnar, P.J. Blower, M.A. Green, R.
T.M. de Rosales, ACS Nano 7 (2013) 500–512.
[17] R.T.M. de Rosales, C. Finucane, J. Foster, S.J. Mather, P.J. Blower, Bioconjug.
Chem. 21 (2010) 811–815.
[18] R.T.M. de Rosales, C. Finucane, S.J. Mather, P.J. Blower, Chem. Commun. (2009)
4847–4849.
[19] R.T.M. de Rosales, R. Tavaré, R.L. Paul, M. Jauregui-Osoro, A. Protti, A. Glaria, G.
Varma, I. Szanda, P.J. Blower, Angew. Chem. Int. Ed. 50 (2011) 5509–5513.
[20] D. Kantoci, J.K. Denike, W.J. Wechter, Synth. Commun. 26 (1996) 2037–2043.
[21] V. Kubicek, J. Rudovsky, J. Kotek, P. Hermann, L.V. Elst, R.N. Muller, Z.I. Kolar, H.
T. Wolterbeek, J.A. Peters, I. Lukes, J. Am. Chem. Soc. 127 (2005) 16477–16485.
[22] Z.X. Jiang, X. Liu, E.K. Jeong, Y.B. Yu, Angew. Chem. Int. Ed. 48 (2009)
4755–4758.
19F MR images were overlayed on to the 1H MR images using
ImageJ software (National Institutes of Health, US). To calculate the
signal to noise ratios (SNR) of the phantom, bladder and liver ROIs
were drawn around the object and also in the background and then
values inputted into the following equation taking into account
[23] E. Bible, F. Dell'Acqua, B. Solanky, A. Balducci, P.M. Crapo, S.F. Badylak, E.T.
Ahrens, M. Modo, Biomaterials 33 (2012) 2858–2871.
[24] C. Giraudeau, B. Djemaï, M.A. Ghaly, F. Boumezbeur, S. Mériaux, P. Robert, M.
Port, C. Robic, D.L. Bihan, F. Lethimonnier, J. Valette, NMR Biomed. 25 (2012)
654–660.
Edelsteins correction factor: SNR = Intensity ROI/(STDEV noise)/
p
(2-p/2) [29].
[25] K.H. Chalmers, A.M. Kenwright, D. Parker, A.M. Blamire, Magn. Reson. Med. 66
(2011) 931–936.
Acknowledgements
[26] F. Schmid, C. Holtke, D. Parker, C. Faber, Magn. Reson. Med. 69 (2013)
1056–1062.
This work was funded by The Centre of Excellence in Medical
Engineering funded by the Wellcome Trust and EPSRC under Grant
No. WT 088641/Z/09/Z. KPS and GDK were funded by the King’s
College London and UCL Comprehensive Cancer Imaging Centre
[27] E.K. Ryu, M. Maccoss, J. Lipid Res. 20 (1979) 561–563.
[28] H.E. Gottlieb, V. Kotlyar, A. Nudelman, J. Org. Chem. 62 (1997) 7512–7515.
[29] P.S. Tofts, The Measurement Process: MR Data Collection and Image Analysis,
Quantitative MRI of the Brain, John Wiley & Sons, Ltd., 2004, pp. 17–54.