Development of PDT/PET Theranostics: Synthesis and Biological Evaluation of an 18F-Radiolabeled Water-Soluble Porphyrin

Article abstract of DOI:10.1021/acs.molpharmaceut.5b00606

Synthesis of the first water-soluble porphyrin radiolabeled with fluorine-18 is described: a new molecular theranostic agent which integrates the therapeutic selectivity of photodynamic therapy (PDT) with the imaging efficacy of positron emission tomograp

Full text of DOI:10.1021/acs.molpharmaceut.5b00606

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Article
Development of PDT/PET Theranostics: Synthesis and Biological
Evaluation of a 18F-Radiolabeled Water Soluble Porphyrin
Guy M. Entract, Francesca Bryden, Juozas Domarkas, Huguette Savoie,
Louis Allott, Stephen J. Archibald, Chris Cawthorne, and Ross W. Boyle
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00606 • Publication Date (Web): 11 Nov 2015
Downloaded from http://pubs.acs.org on November 19, 2015
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Development of PDT/PET Theranostics:
Synthesis and Biological Evaluation of a 18F-
Radiolabeled Water Soluble Porphyrin
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Guy M. Entract , Francesca Bryden , Juozas Domarkas , Huguette Savoie , Louis Allott ,
1,2
1,2
1
3
1
,2
2,4
Stephen J. Archibald , Chris Cawthorne , Ross W. Boyle*
1
1. Department of Chemistry, University of Hull, Cottingham Road, Hull, Yorkshire HU6
RX, UK 2. Positron Emission Tomography Research Centre, University of Hull,
7
Cottingham Road, Hull, HU6 7RX, UK 3. Institute of Cancer Research, 123 Old Brompton
Road, London, SW7 3RP, UK 4. School of Biological, Biomedical and Environmental
Sciences, University of Hull, Cottingham Road, Hull, Yorkshire, HU6 7RX, UK.
+
hv
Photodynamic
Therapy Agent
Therapeutic
1
Theranostic
Agent
O2
γ
Linker
Chain
Diagnostic
PET Imaging
Radionuclide
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Abstract
Synthesis of the first water-soluble porphyrin radiolabeled with fluorine-18 is described; a
new molecular theranostic agent which integrates the therapeutic selectivity of photodynamic
therapy (PDT) with the imaging efficacy of positron emission tomography (PET). Generation
of the theranostic was carried out through the conjugation of a cationic water-soluble
porphyrin bearing an azide functionality to a fluorine-18 radiolabeled prosthetic bearing an
alkyne functionality through click conjugation, with excellent yields obtained in both cold
and hot synthesis. Biological evaluation of the synthesised structures shows both the first
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example of an
F-radiolabeled porphyrin retaining photocytotoxicity following
radiolabeling, as well as demonstrable conjugate uptake and potential application as a
radiotracer in vivo. The promising results gained from biological evaluation demonstrate the
potential of this structure as a clinically-relevant theranostic agent, offering exciting
possibilities for the simultaneous imaging and photodynamic treatment of tumours.
Keywords
Fluorine-18, PET imaging, Photodynamic Therapy, Porphyrins, Theranostics
Introduction
A growing interest in the development of theranostics and “personalised medicine”
highlights the utility of dual-functional theranostic compounds, which combine diagnostic
and therapeutic modalities within the same agent, for the treatment of neoplastic conditions.
Therapeutic strategies exploited to date include nucleic acid delivery, chemotherapy,
hyperthermia (photo-thermal ablation), photodynamic therapy, and radiation therapy, coupled
with imaging strategies including MRI, fluorescent markers, single-photon emission
1
computed tomography (SPECT), and positron emission tomography (PET).
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Porphyrins offer excellent potential as theranostic agents for personalised medicine; their
potent cytotoxic activity within the field of photodynamic therapy has been well
2
documented, demonstrating their increasing clinical relevance in these treatment
3–5
modalities.
In addition, porphyrins demonstrate potential as imaging agents from the
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inherent fluorescence of the tetrapyrrolic core, and also their ability to chelate metals suitable
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for PET, SPECT and MRI (see Boyle et al. for a comprehensive review). PET in particular
offers quantitative imaging of physiological and pathophysiological activities in vivo and is
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well-suited for oncology and whole body imaging.
Some progress has been made towards functionalising PDT agents with positron-emitting
radionuclides for use in PET; however, to date the vast majority of radiolabeling strategies
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have utilised porphyrins as chelating agents for radioisotopes such as Cu,
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68/67
140
Ga, Nd and
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7–12
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(see Waghorn et al. for a comprehensive review of radiolabeled porphyrins).
Ho
Unfortunately, this strategy can inhibit the therapeutic potential of the porphyrin
photosensitiser, as chelation of paramagnetic isotopes quench the photocytotoxic action of
the porphyrin, rendering it useless as a therapeutic agent. These limitations encouraged us to
investigate the attachment of non-metal positron emitting radionuclides onto
photodynamically active porphyrins.
Fluorine-18 is often referred to as the “radionuclide of choice” for PET imaging agents.
The 109.8 minute half-life permits the use of multistep synthesis and allows ample time for
transportation to the clinic, whilst the high proportion of positron decay and relatively low
energy of emitted positrons/positron range facilitate high resolution PET imaging. Despite
this, examples of porphyrins and other tetrapyrrolic photosensitisers radiolabeled with
fluorine-18 in the literature are limited. While methods for fluorine-18 radiolabeling of a
1
porphyrin and a phthalocyanine have been described, neither of these methods are ideal;
4
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both require extended reaction times and harsh reaction conditions, with radiochemical yields
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of less than 25% achieved. In addition, neither demonstrated retention of the cytotoxic action
of the photosensitiser following radiolabeling.
Following our current interest in porphyrin bioconjugation using the Copper (I) –Catalysed
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6–18
Alkyne-Azide Cycloaddition (CuAAC) reaction
, it was rationalised that a click
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conjugation methodology could be utilised to radiolabel a porphyrin under mild reaction
conditions, providing a mild and high yielding method for radiolabeling water-soluble
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porphyrins with minimal impurities.
Results and Discussion
The requirement that we considered essential for designing a porphyrin-fluorine-18 labeled
theranostic agent was that it should combine the activity of both a radiotracer and a
photocytotoxic therapeutic agent. Additionally, the half-life of the chosen radionuclide
dictated that the synthesis be quick and facile, while offering a simple but efficient
purification. The agent was required to be water-soluble, to allow administration without
complex formulation. To date, there have been no reports of fluorine-18 labeled porphyrins
exhibiting these requirements.
Our synthetic route (Scheme 1) utilized a water-soluble cationic porphyrin bearing an azide
clickable moiety, and a PEG chain bearing an alkyne clickable moiety. The fluorine labeled
PEG chain was then clicked to the porphyrin by CuAAC reaction to afford the product.
Porphyrin Synthesis and Characterization
Porphyrin 2 was obtained from the hydrolysis of porphyrin 1 according to a previously
2
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published procedure in 75 % yield. Subsequent synthesis of porphyrin
3
was
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accomplished by diazotization of the amine precursor, with 3 obtained in a yield of 97 %.
Methylation to produce porphyrin 4 was carried out with methyl iodide to produce the water-
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soluble, cationic pyridiniumyl porphyrin 4 giving a 90 % yield, with ammonium
hexafluorophosphate/tetrabutylammonium chloride workup carried out to facilitate anion
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1
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substitution and product purification.
At this stage, zinc chelation of the porphyrin was required to prevent complexation of the
copper catalyst used in the click reaction by the free base porphyrin. While chelation of the
paramagnetic copper catalyst can both interfere with the CuAAC reaction, and quench any
PDT effect of the porphyrin, similar zinc chelated porphyrins have been shown to resist
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copper metallation and also demonstrate potent photocytotoxicity .
º
Scheme 1. (a) propionic acid, 180 C 1 hr, (b) 12 M HCl 100 C 3 hr, then DCM/TEA 9:1 10
º
º
º
2 3 3
min,(c) NaNO soln. and TFA at 0 C 15 min, then NaN soln. added 0 C 1 hr (d) CH I in
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º
DCM 40 C 18h, counter-ion exchange NH
4 6 2
PF and TBAC, (e) Zn(OAc) in water rt 30
mins, then counter-ion exchange NH PF and TBAC.
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Functionalized PEG Chain Synthesis and Characterization
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Two examples of heterobifunctional tetraethylene glycol chains were synthesized to act as
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carriers for F, both functionalized with alkyne groups to allow facile click conjugation to
the photosensitiser. The tetraethoxy PEG chain was selected as a synthetic target due to the
low volatility and excellent amphiphilicity of this structure making it an ideal target for safe
and biocompatible radiolabeling. While 7 was produced with a tosyl group for radiolabeling
via substitution with fluorine-18, fluorine-functionalized 8 was utilized both for radiolabeling
via isotopic substitution and for cold cytotoxicity studies.
º
Scheme 2. (a) NaH in THF at -20 C. Propargyl bromide at rt 18h under N
2
, (b) TsCl, TEA,
º
rt 17 h, (c) anhydrous THF under argon, 80 C,1 M TBAF in THF.
DCM. N
2
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Synthesis of 6 from (OH-PEG
5
-OH) was carried out according to a literature procedure
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to yield 68 % as a pale yellow oil. Tosylation was then carried out to produce 7 in 80 %
1
yield. The synthesis of 8 was initially performed using F in the form of TBAF, with TLC
9
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monitoring showing almost instantaneous generation of 8 from 7 in 90% yield, with product
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Click Conjugation of Porphyrin and Functionalized PEG Chain
To afford synthetic flexibility, initially, click conjugation of 7 and 8 to porphyrin 5 was
attempted, with the aim of producing a porphyrin which could undergo radiolabeling in a one-
step process. However, this direct labelling methodology was found to be unsuitable for two
reasons; in the presence of a source of fluoride ions porphyrin 5 was found to undergo counter
ion exchange on the pyridiniumyl cationic moieties, producing a product with poor water-
solubility and labile fluoride which could easily dissociate in vivo. Secondly, it was found that
under the described conditions, the tosylated PEG chain did not undergo the click reaction. As a
result, click conjugation was then carried out utilising chain 8 only, allowing both cold
optimisation of the click methodology, as well as production of a cold standard of porphyrin
conjugate 9 for both analysis and biological evaluation (Scheme 3).
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Conjugate 9 was synthesized by CuAAC reaction. In contrast to the tosylated chain 7, 8 was
found to undergo rapid and high-yielding click conjugation under the described conditions,
affording the product 9 in excellent yield (90%). Reaction progress was monitored by both TLC
and HPLC; the developed HPLC methodology also provided good separation between the
precursors and final product, allowing for semi-preparative HPLC purification to isolate the
radiolabeled product.
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º
Scheme 3. Copper (II) sulfate, sodium ascorbate and TBTA in 1:3 THF: MW 40 C, 60W, 15
min.
1
8
F labelling of PEG Chain
1
8
Generation of the radiolabeled prosthetic group 8[ F] was achieved via (Scheme 4
nucleophilic substitution of the tosyl group. Initially, reaction of dried fluorine-18 with chain 8
was investigated.
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Scheme 4. (a) dried F, 140 C, 10 min. (b) dried F, 180 C, 10 min.
º
18
º
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was heated with dry F at 180 C for 10 minutes, giving an expected low incorporation of
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.48 % ± 1.37 (n = 3) F (Figure 1), ), with increased heating times showing no significant
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variation. Nucleophilic substitution of 7 demonstrated excellent F labelling, with heating for 10
minutes at 140 ºC resulting in a 93.4 % ± 3.34 (n = 3) radiochemical yield (Figure 2).
Figure 1. RadioTLC of the isotopic substitution reaction, showing <5% incorporation. Region
18
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1: free F, Region 2: incorporated F
Purification of 8 from the precursor 7 was carried out on Sep-Pak®Light tC18 cartridges, to
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remove residual free F, with the product obtained in >99% radiochemical purity. Alternatively,
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generation of the a carrier-added derivative was found to be possible, with residual 7 reacted
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with TBAF to produce a F/ F mixture, giving a single chemical product with no reduction in
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F incorporation observed.
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Figure 2. RadioTLC of the substitution reaction, showing >95% incorporation (left) and >99%
radiochemical purity following purification (right).
Radiolabeled porphyrin conjugate synthesis and characterisation
As previously discussed, under click conditions chain 8 demonstrates excellent conjugation
yields with porphyrin 5, while the precursor 7 shows no reaction with the same porphyrin.
Conveniently, click reaction of mixtures of both 7 and 8 with porphyrin 5 were also found to
yield porphyrin 9 exclusively, and therefore no HPLC purification of the mixture was required.
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CuAAC reaction of the photosensitiser 5 with the radiolabeled chain 8[ F] was performed by
º
heating at 40 C for 20 minutes under previously described conditions. The mixture was purified
on a Sep-Pak® Light tC18 cartridge and washed several times with water before the product was
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eluted with ethanol. Incorporation yields of final product 9[ F] were 34.2 % ± 2.46 (n = 3)
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(
Figure 3).
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Figure 3. RadioTLC of click reaction; 32.8% incorporation of fluorine-18 within 9[ F] (Region
1).
Evaluation of cytotoxicity
Confirmation of the potential of conjugate 9 as a therapeutic agent was carried out through the
evaluation of the photocytotoxicity of the conjugate in a human adenocarcinoma (HT-29) cell
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line. Irradiation of 9 was carried out using an Oriel light system filtered to remove light below
550 nm, and the results were compared to a non-irradiated control. The irradiated light chosen
aligns well with the two Q-bands of the zinc chelated porphyrin, while excluding the Soret band,
giving results which would be applicable to both in vivo testing and clinical use. Human colon
adenocarcinoma cells (HT-29) were incubated for 1 hr with increasing concentrations of 9, and
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-
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irradiated with light (> 550 nm) giving an LD of approximately 1 x 10 M (Figure 4), with
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minimal dark toxicity (>75 % cell survival), observed for all non-irradiated conditions.
Figure 4. Graph of cytotoxicity assay. Assay was carried out on HT-29 cell lines, with results
displayed for conjugate 9 following irradiation (IRR) and non-irradiated control (NI).
Fluorescence and bright field images (Figure 5) of photosensitizer accumulation in the HT-29
cell line were also acquired, demonstrating cellular uptake of the photosensitizer in this cell line.
The bright field image also exhibits blebbing around the cell membrane, indicating rapid onset
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apoptosis due to photosensitizer activation by the excitation source of the microscope, behaviour
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which has previously been reported for cationic porphyrins.
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Figure 5. (Top Image) Fluorescence image showing cellular uptake of photosensitizer 9 in
human adenocarcinoma (HT-29) cells, (Bottom Image) Bright field image of HT-29 cells
incubated with 9.
In vivo radiochemistry
18
18
Preparation of the 8[ F] and subsequent click reaction to form conjugate 9[ F] was performed
1
as described in the experimental, with both crude 8[ F] and 9[ F] purified by semi-preparative
8
18
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HPLC after each stage of the reaction. Fluorination of 8[ F] was carried out in acetonitrile, to
yield the product in 77 % RCY (non-decay corrected) and >99% RCP by HPLC. Click reaction
1
to the porphyrin was then carried out at 60°C to produce 9[ F] in 16 % RCY (non-decay
8
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corrected) and >97 % RCP by HPLC. The isolated sample of 9[ F] demonstrated good specific
activity of 1GBq/µmol, with an obtained log P value of -2.48 (PBS:octanol).
18
Figure 6 . RadioTLC of the nucleophilic substitution synthesis of 8[ F], showing >75 %
incorporation.
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Figure 7. RadioTLC of click reaction synthesis of 9[ F], with over 66% incorporation
8
demonstrated.
In vivo evaluation
18
Tumour uptake was assessed in the HT29 and U87 xenograft models. Conjugate 9[ F] was
1
8
injected intravenously (i.v.) and biodistribution was quantified by PET imaging (Dynamic 9[ F]
uptake in tumour bearing animals. A and B: Transaxial and coronal image slices centred on the
tumour at 80-90 minutes, showing uptake in tumour and liver in representative HT29 tumour
18 18
arrows). C, D: Time activity curves of 9[ F] in liver E,F: Time activity curves of 9[ F] in
(
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tumour and muscle. All time activity curves represent average data from four tumour-bearing
animals ±S.E.M.8).
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Figure 8. Dynamic 9[ F] uptake in tumour bearing animals. A and B: Transaxial and coronal
image slices centred on the tumour at 80-90 minutes, showing uptake in tumour and liver in
18
representative HT29 tumour (arrows). C, D: Time activity curves of 9[ F] in liver E,F: Time
18
activity curves of 9[ F] in tumour and muscle. All time activity curves represent average data
from four tumour-bearing animals ±S.E.M.
1
As expected, 9[ F] accumulated mainly in the liver (Figure 8 C&D), consistent with previously
8
13
reported data on porphyrins . There was also evidence of tumour accumulation in both models,
with average tumour-to-muscle ratios of 1.4 (SUVmean) or 2.3 (SUVmax) at 80-90 minutes
(Figure 8 E&F).The accumulation of untargeted porphyrins in tumour tissue is thought to be due
to the enhanced permeability and retention (EPR) effect, and this is assumed to be high in
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preclinical xenograft models . For the purposes of this study we assessed uptake in HT29 and
U87 tumours, as these have been reported to to effect low or high levels of EPR mediated
2
7,28
nanoparticle accumulation respectively
. Tumour uptake was observed in all tumours,
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exhibiting the potential of this theranostic agent for calculating the optimal irradiation time for
clinical PDT treatment.
Conclusions
In conclusion, we have demonstrated that a single theranostic agent in the form of a novel
18
fluorine-18 radiolabeled water-soluble porphyrin can be synthesized from an alkyne-bearing F
18
PEG carrier chain and an azide functionalised porphyrin. This is the first demonstration of an F
radiolabeled porphyrin retaining photocytotoxicity following radiolabeling; with the synthesised
compound showing cellular uptake, good photocytotoxicity and minimal dark toxicity in a
relevant human cancer cell line, as well as confirmed uptake of the conjugate into neoplastic
tissue and demonstrated potential as a radiotracer in vivo. The promising results gained from
biological evaluation demonstrate the potential of this structure as a clinically-relevant
theranostic agent, offering exciting possibilities for the simultaneous imaging and photodynamic
treatment of tumours.
Experimental
Materials and methods
1
13
H and C NMR spectra were recorded on JEOL Eclipse 400 and JEOL Lambda 400
1
13
spectrometers (operating at 400 MHz for H and 100 MHz for C). Chemical shifts (δ) are
reported in parts per million (ppm), referenced to CDCl and DMSO. Coupling con- stants ( J)
3
are recorded in Hz and significant multiplicities described by singlet (s), doublet (d), multiplet
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(
m). MALDI mass spectra were performed by the EPSRC National Mass Spectrometry
Facilities, Swansea, UK. UV–visible spectra were recorded on a Varian Cary spectrophotometer.
Chemical reagents and solvents were purchased from Sigma-Aldrich and Alfa Aesar, and were
used as received unless otherwise stated. Microwave reactions were carried out in a CEM
Discover Benchmate microwave reactor controlled using Synergy software. In all cases,
maximum stirring, maximum pressure of 200 bar and 2 minute maximum heating step were
used. Reaction temperatures were monitored using an external IR probe and carried out in a 10
ml sealed reaction vessel. Irradiation of cells during phototoxicity assays was carried out using
an Oriel lamp system with a Schott 06515 Long Pass Optical Filter, allowing irradiation with 95–
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18
18
8% of light above 515 nm. Fluorine-18 ( F) was produced by the 18O(p, n) F nuclear
9
reaction using a 7.5 MeV ABT Biomarker Generator cyclotron (ABT Molecular Imaging, Inc.©,
18
USA) and [ O]H O >98% (CMR©, Russia), being the current reaching the target ~4.2 µA.
2
1
8
Specific activity of the produced F was an average of 100 GBq.mol-1, according to the
manufacturer. Analytical HPLC was performed through an Agilent 1200 series with a 254 nm
UV detector (Agilent Technologies©, USA) and effluent radioactivity was monitored using a
NaI crystal coupled to a photomultiplier tube (LabLogic Systems Limited©, UK). Purification by
semi-preparative HPLC was performed through an Agilent 1100 series with a 254 nm UV
detector (Agilent Technologies©, USA) and effluent radioactivity was monitored using a NaI
crystal coupled to a photomultiplier tube (LabLogic Systems Limited©, UK). An ACE 5 C18
1
0*250 5A column was used, with all products eluted eluted using MeCN:water gradients
containing 0.1% TFA (flow rate 4.7 ml/min).
Synthetic procedures
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Synthesis of 4: Methyl iodide (2 ml, 32.3 mmol) was added via syringe to a stirred solution of
5
-[4- azidophenyl]-10,15,20-tri-(4-pyridyl) porphyrin (100 mg, 0.151 mmol) in dimethyl
º
formamide (DMF) (10 ml). The reaction was then stirred at 40 C overnight. After filtering
through cotton wool, the residue was dissolved in water. Ammonium hexafluorophosphate was
added, and the product filtered. The solid was dissolved in acetone and tetrabutylammonium
chloride was added, and the mixture filtered. The crude product was precipitated from diethyl
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ether over MeOH to yield a dark brown solid (148 mg, 90%). UV-vis (H O): max, 424, 520, 562,
2
-
1
-1 1H
590, 648. log (424 nm) = 5.11 M cm . -NMR (DMSO-d6): δ 4.74 (s, 9H, CH ),7.65 (d, 2H, 5-
3
m-Ph), 8.28 (d, 2H,5-o-Ph), 9.01 (m, 6H, 10,15,20-o-Py), 9.12 (m, 8H, βH), 9.07 (m, 6H,
1
3
1
0,15,20-m-Py). C-NMR (DMSO-d6): δ 40.13 (CH ), 114.66, 115.42, 117.63, 122.87, 132.11,
3
132.25 (β-C), 135.53, 143.62 (β-C), 147.88, 148.15, 148.35, 150.19, 158.54. MS: (ESI) m/z 351
100[M -3Cl] 2+), HRMS: calcd. For C H N : 351.1478 found 351.1481.
(
4
6
33 10
Synthesis of 5: Porphyrin 4 (10.0 mg 0.0141 mmol) was dissolved in water (10 ml) and
zinc(II) acetate (3.00 mg, 0.0133 mmol) added. The mixture was stirred at room temperature for
hour and checked for completion by TLC. Ammonium hexafluorophosphate was added, and
1
the product filtered. The solid was dissolved in acetone and tetrabutylammonium chloride was
added, and the mixture filtered. The crude product was precipitated from diethyl ether over
1
MeOH (10.4 mg, 95 %). UV-vis (H O): max, 436, 564, 612. H-NMR (DMSO-d6): δ 4.72 (s,
2
9H, CH ),7.61 (d, 2H, 5-m-Ph), 8.21 (d, 2H,5-o-Ph), 8.9-9.06 (m, 14H, 10,15,20-o-Py, βH), 9.44
3
13
(
m, 6H, 10,15,20-m-Py). C-NMR (DMSO-d6): δ 48.26 (CH -N ), 115.26, 116.03, 118.21,
2
3
1
22.73, 132.13, 132.65(β-C), 133.70, 136.13, 139.44, 139.87, 144.22(β-C), 148.75, 148.92,
150.75, 159.10. MS: (ESI) m/z 400 (100[M -2Cl] 2+), HRMS: calcd. For C H Cl N Zn :
4
4
33
1
10
1
4
00.0929 found 400.0932.
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Synthesis of 6: Tetraethylene glycol (2.70 g, 13.9 mmol) was slowly added to a solution of
º
NaH (60 % in mineral oil, 0.8 g, 20 mmol) in THF (20 ml) at -20 C. Propargyl bromide (80 % in
toluene, 1.48 ml, 13.9 mmol) was added after all bubbling stopped and the mixture was stirred
overnight at room temperature and nitrogen. The mixture was filtered and solvent removed under
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reduced pressure. The crude was then purified by column chromatography (4.5 % MeOH :
1
DCM) to yield the product as a pale yellow oil (1.82 g, 68%). H-NMR (CDCl ): δ 2.40 (s, 1H,
3
C≡CH), 2.92 (OH), 3.52-3.66 (m, 16H), 4.13 (d, 2H, CH -C).13C-NMR- (CDCl ): δ58.39 (CH -
2
3
2
C), 61.68 (CH -OH), 69.13, 70.37, 70.41, 70.56, 70.58, 70.65, 72.61, 74.65 (C≡CH), 79.66
2
(C≡CH). MS: (ESI) m/z 233 (100[M + H]+), HRMS: calcd. for C11H21O5: 233.1384 found
233.1380
Synthesis of 7: TsCl (1.60 g, 8.39 mmol) and TEA (1.17 ml, 8.39 mmol) were added to a
stirred solution of PEG 6 (1.86 g, 8.00 mmol) in DCM. The mixture was stirred under nitrogen at
room temperature for 17 hours. The solvent was removed under reduced pressure and the
product purified by column chromatography to yield the product as a pale yellow oil (2.47 g, 80
1
%). H-NMR (CDCl ): δ 2.38 (s, 4H, C≡CH, CH ), 3.45-3.65 (m, 14H), 4.02-4.14 (m, 4H, CH -
3
3
2
1
3
C, CH ), 7.26 (d, 2H, J=7.9 Hz), 7.70 (d, 2H, J=7.1 Hz). C-NMR (CDCl3): δ21.57 (CH ),
2
3
5
8.41 (CH -C), 68.75, 69.09, 69.40, 70.35, 70.54, 70.66, 70.73, 74.76 (C≡CH), 79.73 (C≡CH),
2
127.98, 129.93, 130.37, 132.95 (4-Ar), 144.92 (1-Ar).MS: (ESI) m/z 404 (100[M + NH4] +),
HRMS: calcd. For C H O N S : 404.1737 found 404.1726.
1
8
30
7
1 1
Synthesis of 8: PEG 7 (2.47 g, 6.40 mmol) was dissolved in 10 ml of anhydrous THF under
º
argon. The solution was heated to 80 C and a solution of 1 M TBAF in THF (4.84 ml, 4.84
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mmol) was added. The product was allowed to cool and the solvent was removed under reduced
pressure. The product was purified by column chromatography using (25 % DCM : ethyl acetate)
1
to give a pale yellow oil (1.35 g, 90 %). H-NMR (CDCl ): δ 2.40 (s, 1H, C-CH), 3.58-3.68(m,
3
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4H), 4.14 (d, 2H, O-CH -C), 4.5, 48 Hz, 4 Hz(dt, 2H, F-CH ). C-NMR (CDCl ): δ21.57
2
2
3
(CH ), 58.40 (CH -C), 70.39, 70.59, 70.69, 70.78, 74.62(C≡CH), 79.73 , 82.38, 84.05, 128.97 J
3
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=
744 Hz(CH2-F). F-NMR (CDCl ): δ 179.50.MS: (ESI) m/z 252 (100[M + NH ] +), HRMS:
3
4
calcd. For C H F O N : 252.1606 found 252.1607.
1
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4
Synthesis of 9: Porphyrin 5 (10.0 mg 0.0118 mmol) was added to a 10 ml microwave tube and
PEG 8 (3.00 mg, 0.013 mmol) in THF : water (1 : 3, 8 ml) was added. Copper (II) sulphate
pentahydrate (5.00 mg, 0.0200 mmol), sodium ascorbate (5.00 mg, 0.0250 mmol) and TBTA
º
1.00 mg, 1.90 mmol) were added and the mixture heated to 45 C by microwave (75 W, max
(
pressure 100 bar) for 20 minutes. The mixture was concentrated under reduced pressure and
ammonium hexafluorophosphate was added. The mixture was filtered and redissolved in
acetone. Tetrabutylammonium chloride was added and the mixture filtered. The product was
precipitated from diethyl ether over MeOH to yield the product as a green solid (11.4 mg, 90 %).
-
1
-1 1
UV-vis (H O): max, 435, 565, 614 log (435 nm) = 5.03 M cm . H-NMR (DMSO-d ): δ 3.65
2
6
(m, 14H), 4.53, J=24 Hz,4 Hz (dt,2H, CH -F),4.72 (s, 9H, CH ), 4.77 (s, 2H, O-CH -Triazole),
2 3 2
8.34-8.44 (m, 4H, 5-Ph), 8.85-9.06 (m, 14H, 10,15,20-m-Py, m 8H, βH), 9.20 (s, 1H, Triazole-
13
H), 9.44 (d, 6H, 10,15,20-o-Py). C-NMR (DMSO-d ): δ 40.13, 48.26, 58.05, 64.06, 69.77,
6
70.44.MS: (ESI) m/z 333 (100[M -3Cl] 3+), HRMS: calcd. For C H O N F Zn : 333.1144
55 52 4 10 1 1
found 333.1152.
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Radiochemistry
Fluorine production: The fluorine-18 radionuclide was produced by the O(p,n) F reaction on
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an enriched water target. Oxygen-18 water containg 80 MBq of F was transferred to a 5ml V-
vial containing potassium carbonate solution in water (0.1M, 200 ꢀl), and Kryptofix K222 (5 ug)
in water (300 ꢀl). The water was removed via azeotropic distillation with acetonitrile under a
º
compressed air stream at 110 C.
18
Synthesis of 8[ F] via isotopic substitution: To a 5ml V-vial containing the dried 18F was
º
added 8 (5 mg, 0.021 mmol) in dry DMSO (200 ꢀl) and the mixture heated to 180 C for 10
minutes. The mixture was diluted with methanol (1 ml) and passed through a Sep-Pak Light
Alum N cartridge. Labelling yields were determined by radioTLC, and the mixture was used
without further purification.
18
Synthesis of 8[ F] from 7: To a 5ml V-vial containing the dried 18F was added 7 (5 mg,
º
0
.013 mmol) in dry DMSO (200 ꢀl) and the mixture heated to 140 C for 10 minutes. The
mixture was diluted with methanol (1 ml) and passed through a Sep-Pak Light Alum N cartridge.
Labelling yields were determined by radioTLC, and the mixture was used without further
purification.
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Synthesis of carrier-added 8[ F]: To a 5ml V-vial containing the dried F was added 7 (5 mg,
º
0
.013 mmol) in dry DMSO (200 ꢀl) and the mixture heated to 140 C for 10 minutes.
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Tetrabutylammonium fluoride (5 mg, 0.019 mmol) in dry DMSO (200 ꢀl) was added and the
º
mixture heated to 140 C for 5 minutes The mixture was diluted with methanol (1.5 ml) and
purified on a Sep-Pak Light Alum N cartridge. Labelling yields were determined by radio-TLC,
and the mixture was used without further purification.
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General methodology for click reaction 9[ F]: To a 10 ml microwave tube containing 8[ F]
was added 5 (1 mg, 1.1 ꢀmol) in water (0.2 ml) and a solution of TBTA (174 ꢀg, 0.4 ꢀmol), and
copper (II) sulfate pentahydrate (50 ꢀg, 0.2 ꢀmol) and sodium ascorbate (80 ꢀg, 0.4 ꢀmol) in
º
water (0.2 ml). The mixture was heated to 40 C for 20 minutes (MW, 60 W). The mixture was
diluted with water (20 ml) and passed through a Sep-Pak Light C18 cartridge. The cartridge was
washed with water (5 ml) and the product eluted with ethanol (0.5 ml).
in vivo Radiochemistry
18
Fluorine production: Oxygen-18 water (~140 ꢀL) containing 1.8 GBq of F was transferred to a
5
ml V-vial containing potassium hydrogencarbonate solution (0.5 ml, 5 mg/ml) and Kryptofix
K222 (10.2 mg in 1 ml of dry acetonitrile). The water was removed via azeotropic distillation
º
with acetonitrile under a compressed argon stream at 90 C.
18
Synthesis of 8[ F]: To a 5 ml V-vial containing dried 18F (1.4 GBq) was added 7 (6.7 mg) in
dry acetonitrile (1 ml) and the mixture heated to 90 °C for 10 minutes. The crude reaction
mixture was passed through Sep-Pak Light Alum N cartridge. Eluted activity: 982 MBq, (RCY
o
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residue diluted with water (0.5 ml). The crude was purified on semi-preparative HPLC
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MeCN:water, 20% to 30% in 15 min), with 8[ F] eluting at 10-11 min (726 MBq). The
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collected HPLC fraction was diluted to 40 ml with water and loaded on home-made Oasis C18
Lite Sep-Pak cartridge, washed with water (10 ml) and eluted with methanol (2 ml). Methanol
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solution containing activity was dried at 60 C.
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Synthesis of 9[ F]: To a 1.7 ml champagne HPLC vial containing pure 8[ F] (446 MBq) was
added a combined solution of copper (II) sulfate pentahydrate (0.5 mg in 12.5 µl water), sodium
ascorbate (3.75 mg in 50 µl water), TBTA (0.5 mg in 160 µl methanol) and porphyrin (0.2 mg
in 100 µl water). The reaction mixture was heated to 60 °C for 35 minutes. The crude product
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was purified on semi-preparative HPLC (MeCN:water, 20% to 40% in 30 min) with 9[ F]
eluting at 15-16 min (72.6 MBq, RCY 16.2 %). The fraction was diluted with water and purified
on an Oasis C18 Sep-Pak cartridge. The product was eluted with ethanol (0.5 ml) while filtering
on a 0.22 µm filter. The solution was evaporated to dryness under stream of compressed argon at
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0 C and dissolved in 0.2 uL of heparin-water (33.6 MBq).
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Quality control
All reactions were monitored, and incorporation was determined by radio-TLC using silica TLC
sheets, in 9:1 DCM/MeOH solution. RCY and RCP was determined for all compounds using
analytical HPLC under gradient conditions (MeCN:water with 0.1 % TFA, 25-40 % over 30
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mins). The compounds were eluted after the following retention times (tR): 8[ F] 8.5 min,
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[ F] 16:6 min.
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[ F] (3.4 MBq) was partitioned in a mixture of 1 ml of 1-octanol and 1 ml of PBS after being
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vortexed for 5 min. Following centrifugation at >1,200 g for 5 min, the octanol and aqueous
phases were sampled and counted. Reported log P value is based on the average of three
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PET imaging
CD-1 nu/nu female mice (Charles River, UK) bearing subcutaneous HT29 or U87 xenografts of
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were placed in the Minerve Small-Animal Imaging Cell (Minerve, France) and transferred to the
SuperArgus 2R preclinical PET/CT scanner (Sedecal, Spain). At the start of the PET acquisition,
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animals were injected with ~10MBq of F-9 intravenously (IV) via the tail vein and 90-minutes
of data were acquired using the 250-700 keV energy window. Data was histogrammed into 3 x
120, 1 x 240, 4 x 600 and 2 x 1200-second bins and reconstructed into a 175 x 175 x 61 (0.3875
x 0.3875 x 0.775mm) matrix using OSEM2D with 16 subsets and 2 iterations and
randoms/scatter/attenuation correction via Sedecal MMWKS software. The image data was
normalized to correct for nonuniformity of response, dead-time count losses and radioactive
decay to the time of injection. Activity concentrations were determined by conversion of count
rate data from reconstructed images. Regions of interest were drawn around the tumour and
sections of quadriceps muscle and liver, and time-activity curves generated using AMIDE
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software (Loening & Gambhir, Molecular Imaging 2003). CT images were acquired via 360
projections/8 shots at a tube voltage of 45kV and current of 140µA. Respiration and temperature
were monitored throughout using the SA Instruments 1025T monitoring system (SA Instruments,
CA, USA). All procedures were carried out in accordance with the Animals in Scientific
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Procedures Act 1986 and UKCCCR Guidelines 2010 by approved protocols following
institutional guidelines (Home Office Project License number 60/4549 held by Dr. Cawthorne).
Author Information
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Corresponding Author
*E-mail R.W.Boyle@hull.ac.uk. Phone: +44 [1482466353]
Author Contributions
The manuscript was written with contributions from all authors, with approval upon final
version.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
We are grateful to Gonçalo Clemente for production of fluorine-18. Mass spectrometry data
was acquired at the EPSRC UK National Mass Spectrometry Facility at Swansea University.
Supporting Information
Video of overall biodistribution over time in mouse model.
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