Efficient radiosynthesis and preclinical evaluation of [18F]FOMPyD as a positron emission tomography tracer candidate for TrkB/C receptor imaging

Article abstract of DOI:10.1002/jlcr.3827

Herein we report an efficient radiolabeling of a ¹⁸F-fluorinated derivative of dual inhibitor GW2580, with its subsequent evaluation as a positron emission tomography (PET) tracer candidate for imaging of two neuroreceptor targets implicated in the pathophysiology of neurodegeneration: tropomyosin receptor kinases (TrkB/C) and colony stimulating factor receptor (CSF-1R). [¹⁸F]FOMPyD was synthesized from a boronic acid pinacolate precursor via copper-mediated ¹⁸F-fluorination concerted with thermal deprotection of the four Boc groups on a diaminopyrimidine moiety in an 8.7±2.8% radiochemical yield, a radiochemical purity >99%, and an effective molar activity of 187±93 GBq/μmol. [¹⁸F]FOMPyD showed moderate brain permeability in wild-type rats (SUV_max = 0.75) and a slow washout rate. The brain uptake was partially reduced (ΔAUC_40–90 = 11.6%) by administration of the nonradioactive FOMPyD (up to 30 μg/kg). In autoradiography, [¹⁸F]FOMPyD exhibits ubiquitous distribution in rat and human brain tissues with relatively high nonspecific binding revealed by self-blocking experiment. The binding was blocked by TrkB/C inhibitors, but not with a CSF-1R inhibitor, suggesting selective binding to the former receptor. Although an unfavorable pharmacokinetic profile will likely preclude application of [¹⁸F]FOMPyD as a PET tracer for brain imaging, the concomitant one-pot copper-mediated ¹⁸F-fluorination/Boc-deprotection is a practical technique for the automated radiosynthesis of acid-sensitive PET tracers.

Full text of DOI:10.1002/jlcr.3827

Kostikov Alexey (Orcid ID: 0000-0003-1646-761X)
Efficient radiosynthesis and preclinical evaluation of [18F]FOMPyD as a PET tracer
candidate for TrkB/C receptor imaging.
Abbreviated title: [18F]FOMPyD radiosynthesis and preclinical evaluation as a PET
tracer.
Thomas A. Singleton,1 Hussein Bdair,1,4 Justin J. Bailey,2 Sangho Choi,2 Arturo Aliaga,3 Pedro
Rosa-Neto,1, 3, 4 Ralf Schirrmacher,2 Vadim Bernard-Gauthier2 and Alexey Kostikov1,4
1
McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University,
Montreal, QC, H3A 2B4, Canada.
2
Department of Oncology, Division of Oncological Imaging, University of Alberta,
Edmonton, AB, T6G 2R3, Canada.
3 Translational Neuroimaging Laboratory, McGill Centre for Studies in Aging, Douglas Mental
Health University Institute, Montreal, QC, H4H 1R3, Canada
4
Department of Neurology and Neurosurgery, McGill University, Montreal, QC, H3A 2B4,
Canada.
Keywords: Brain imaging, positron emission tomography, PET, GW2580, [18F]FOMPyD,
fluorine-18, copper-mediated 18F-fluorination, tropomyosin receptor kinase (Trk), colony
stimulating factor receptor (CSF-1R).
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jlcr.3827
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18
Abstract. Herein we report an efficient radiolabeling of a F-fluorinated derivative of dual
inhibitor GW2580, with its subsequent evaluation as a positron emission tomography (PET)
tracer candidate for imaging of two neuroreceptor targets implicated in the pathophysiology of
neurodegeneration: tropomyosin receptor kinases (TrkB/C) and colony stimulating factor
receptor (CSF-1R). [18F]FOMPyD was synthesized from a boronic acid pinacolate precursor
via copper-mediated 18F-fluorination concerted with thermal deprotection of the four Boc
groups on a diaminopyrimidine moiety in an 8.7±2.8% radiochemical yield, a radiochemical
purity >99% and an effective molar activity of 187±93 GBq/µmol. [18F]FOMPyD showed
moderate brain permeability in wild-type rats (SUVmax = 0.75) and a slow washout rate. The
brain uptake was partially reduced (ΔAUC40–90 = 11.6%) by administration of the non-
radioactive FOMPyD (up to 30 μg/kg). In autoradiography, [18F]FOMPyD exhibits ubiquitous
distribution in rat and human brain tissues with relatively high non-specific binding revealed
by self-blocking experiment. The binding was blocked by TrkB/C inhibitors, but not with a
CSF-1R inhibitor, suggesting selective binding to the former receptor. Although an unfavorable
pharmacokinetic profile will likely preclude application of [18F]FOMPyD as a PET tracer for
brain imaging, the concomitant one-pot copper-mediated 18F-fluorination/Boc-deprotection is
a practical technique for the automated radiosynthesis of acid-sensitive PET tracers.
Introduction. Tropomyosin receptor kinases (TrkA/B/C) are part of the large family of
tyrosine kinases, which regulate neuronal differentiation, growth, and survival via their
interactions with four neurotrophin factors.1 Downregulation of Trk has been reported in many
pathological conditions of the central nervous system (CNS), including Alzheimer’s disease
(AD) and other neurodegenerative conditions.2 In addition, tyrosine kinase inhibitors (TKI) are
considered one of the most promising anticancer treatments3 due to upregulation and increased
activity of Trk associated with several types of cancer.4 Colony stimulating factor receptor
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(CSF-1R) belongs to class III of the tyrosine kinases, called the platelet-derived growth factor
(PDGF) receptor family. In the CNS, CSF-1R is almost exclusively expressed on microglia
cells and is crucial for their viability.5 Thus, CSF-1R inhibitors have been suggested as
potential modulators of microglial activity in neurodegenerative diseases.6,7 Imaging tools to
further study expression and function of both Trk and CSF-1R in the living brains and predict
outcomes of the receptor activity-mediating therapies are highly sought after.
Recently, several positron emission tomography (PET) tracers based on type-I TrkB/C
inhibitors have been developed and evaluated in preclinical and clinical settings.8 In particular,
[11C]IPMICF169 and [18F]TRACK10 (Figure 1, A) were the first type-I TKI-based PET tracers
used for endogenous kinase neuroimaging in non-human primates and humans.11 Two putative
selective tracers for in vivo imaging of CSF-1R have been reported to date as attractive
candidates for the PET imaging of microglial activation beyond translocator protein (TSPO)12
(Figure 1, B). [11C]CPPC showed elevated brain uptake in animal models of
neuroinflammation and AD in vivo, and increased binding to post-mortem human brain tissues
of AD patients in autoradiography.13 Although [11C]AZ683 has poor brain permeability in
rodents and nonhuman primates, it was suggested as a potential tracer for peripheral imaging
of inflammation.14
The 18F-fluorinated analog of dual type-II Trk/CSF-1R inhibitor GW258015 5-(4-((4-
[18F]fluorobenzyl)oxy)-3-methoxybenzyl)pyrimidine-2,4-diamine ([18F]FOMPyD, Figure 1,
C) was the first reported putative dual Trk/CSF-1R tracer.16 However, a cumbersome and low-
yielding 4-step radiosynthesis was necessary due to the lack of radiochemistry tools available
at the time, which precluded evaluation of this tracer as an imaging probe. In light of recent
advances in Trk and CSF-1R PET tracer developments, there was a great interest in evaluating
[18F]FOMPyD via PET imaging and autoradiography.17 As a type-II Trk inhibitor, this PET
tracer candidate was hypothesized to display improved pharmacokinetics compared to Trk
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tracers based on type-I inhibitors due to reduced endogenous competition with ATP.18 In the
last 5 years, several methodologies developed to enable late-stage incorporation of fluorine-18
in non-activated aromatic rings have significantly expanded the scope of synthetically-
accessible PET tracers. In this work we aimed to develop an efficient radiosynthesis of
[18F]FOMPyD via the copper-mediated 18F-fluorination of boronic acid esters, reported
independently by the groups of Gouverneur19 and Scott,20 and evaluate it as an imaging probe
by preclinical PET imaging and autoradiography in both animal and human brain tissues.
Experimental. The synthesis of boronic acid pinacolate precursor 6 was devised based on the
modified previously reported synthesis of the original ligand FOMPyD. Briefly, the reaction
of p-iodobenzyl bromide with vanillin affords 1, and the subsequent condensation with 3-
morpholinopropionitrile followed by exchange of the morpholino group with aniline, and
subsequent cyclization with guanidine yields 4. The diaminopyrimidine moiety is then
protected by the DMAP-catalyzed installation of four tert-butyloxycarbonyl (Boc) groups (5),
which allow the subsequent Miyaura borylation (6) and final radiolabeling reaction to proceed
without interference from the free amines. Precursor 6 was synthesized in 38% overall yield
starting from p-iodobenzyl bromide according to the conditions outlined in Figure 2.
For the isolation of 18F-fluoride from the target [18O]H2O we applied the “minimalistic
approach” originally reported by Zischler et al.,21 which proved to be the most effective
technique for the copper-mediated synthesis of other 18F-labeled tracers in our hands.10 Briefly,
[18F]F−/H2O was passed through a Sep-Pak Light QMA cartridge (46 mg, Waters) followed by
anhydrous methanol (3 mL) to remove traces of water. 18F-fluoride was then eluted in the
opposite direction with tetraethylammonium bicarbonate (TEAB) solution in methanol (450
μL, 1 mg/mL) followed by neat methanol (500 µL) into a reaction vial and the solvent was
removed at 90 °C under vacuum and a sweep stream of argon.
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In radiolabeling optimization experiments, a solution of 6 (8.6 mg, 10 µmol) and
Cu(OTf)2(Py)4 (6.8 mg, 10 µmol) in a DMA/1-BuOH solvent mixture (2:1, 450 µL total) was
added to the dried [18F]TEAF/TEAHCO3 and the reaction mixture was heated stepwise to
110 °C and then to 130 °C with monitoring by radioTLC. After 10 min at 110 °C we observed
multiple (≥ 4) radioactive spots attributed to different partially-deprotected 18F-fluorinated
species, which started to merge after an additional 10 min heating at 110 °C and fully converted
into a single spot corresponding to [18F]FOMPyD (RCC of 26%, Figure S3) after heating the
reaction mixture to 130 °C for an additional 10 min. Following initial optimization, the
shortened radiosynthesis (10 minutes at 110 °C followed by 10 minutes at 130 °C) was
automated using a radiosynthesis unit (Scintomics GRP) equipped with a semi-preparative
HPLC module (Knauer). [18F]FOMPyD was purified on a Phenomenex Luna® C18(2) column
(10 µm, 250 × 10 mm), using a mobile phase of acetonitrile/20 mM aqueous NaH2PO4 (30:70)
at a flow rate of 4 mL/min. The tracer was then reformulated in 10% aqueous ethanol and its
identity was confirmed by coinjection with authentic FOMPyD on an analytical HPLC system
(Agilent 1200) equipped with a Phenomenex Prodigy ODS-3 column (250 × 4.6 mm, 10 μm),
using a mobile phase of acetonitrile/0.1% aqueous TFA (40:60) at a flow rate of 0.7 mL/min
(Figure S1). The molar activity was calculated by quantification of the UV signal using the
calibration curve (Figure S2).
18
Results and discussion. In previous reports the copper-mediated F-fluorination of amines
required an acid-catalyzed Boc-deprotection in concentrated HCl21 or HI22 at 130 °C as the
second step, which would not be suitable for many even mildly acid-sensitive compounds. We
aimed to apply a concomitant thermal cleavage of the four Boc protecting groups to avoid acid-
catalyzed decarboxylation. This methodology has been used for PET tracers synthesized via
conventional SNAr 18F-fluorination, such as [18F]T807 (now [18F]AV1451)23 and
18
[18F]MK6240.24 However, the concerted Cu-mediated F-fluorination of boronic acid esters
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with Boc deprotection has not been previously reported to the best of our knowledge. This was
achieved by a stepwise (110 °C → 130 °C) heating procedure as described above.
Starting from 2.6–13.1 GBq of [18F]F−, [18F]FOMPyD was reliably obtained in an 8.7±2.8%
decay-corrected radiochemical yield (RCY), an effective molar activity (Am) of 187±93
GBq/µmol and with a radiochemical purity >99% (n = 4). Importantly, the tracer was free from
the unreacted 6 and other non-radioactive impurities, including the product of the
protodeboronation reaction 5-(4-(benzyloxy)-3-methoxybenzyl)pyrimidine-2,4-diamine
(BOMPyD, Figure 3), a known major side-product in this reaction25 (Figure S1). This
procedure represents a major improvement over the previously-reported four-step sequence
and, for the first time, allowed for preclinical evaluation of [18F]FOMPyD by in vitro
autoradiography in human and rat post-mortem brain tissues and in vivo PET imaging in wild-
type rats.
In baseline in vitro autoradiography experiments on rat brain tissues, [18F]FOMPyD exhibited
ubiquitous binding in all regions. Homologous blocking revealed modest binding specificity as
the signal was blockable by the non-radioactive FOMPyD (100 µM) across all analyzed regions
(p < 0.05), particularly in all cortical regions (Δ = 37.4%, p < 0.001), as shown in Figure 4, A.
To further investigate the binding selectivity, we performed heterologous blocking studies on
rat and human brain tissues against a panel of kinase inhibitors, including the
protodeboronation side product BOMPyD, type-I pan-Trk inhibitor (R)-IPMICF22 (TrkB/C
IC50 = 0.57/0.37 nM, respectively),9 type-II pan-Trk inhibitor PF-06273340 (TrkA/B/C IC50 =
6/4/3 nM, respectively)26 and CSF-1R inhibitor BLZ945 (IC50 = 1.2 nM).27 Across all rat brain
cortical regions (posterior, medial and anterior cortices; Figure 5, A) blocking with BOMPyD,
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a demethoxy analogue of dual Trk/CSF-1R inhibitor GW2580, was the highest (Δ = 42.1 ±
6.8%; t(28) = 10.574, p < 0.001; n = 15). Among selective Trk inhibitors, PF-06273340 showed
statistically significant blocking (Δ = 13.2 ± 9.7%; t(28) = 2.460, p < 0.05; n = 15), while (R)-
IPMICF22 reduced tracer binding with lower efficiency and statistical significance (Δ = 10.9
± 15.9%; t(28) = 1.948, p = 0.06; n = 15). Notably, blocking with BLZ945 (Δ = −0.1 ± 8.6%;
t(28) = 0.010, p = 0.99; n = 15) did not result in any significant reduction in binding of
[18F]FOMPyD. In human brain cortical tissues (posterior cingulate cortex, PCC; Figure 5, B)
we observed a very similar blocking pattern. BOMPyD remained the most efficient blocking
agent (Δ = 41.1 ± 17.3%; t(6) = 3.170, p < 0.05; n = 4), while blockage with PF-06273340 (Δ =
30.6 ± 17.8%; t(6) = 2.529, p < 0.05; n = 4) was more efficient compared to the rat brain tissues.
(R)-IPMICF22 (Δ = 24.2 ± 10.7%; t(6) = 1.958, p = 0.098; n = 4) also appeared to reduce tracer
binding more efficiently, however the blocking remained not statistically significant, possibly
due to the limited sample size. Finally, BLZ945 (Δ = 3.2 ± 3.3%; t(6) = 0.332, p = 0.75; n = 4)
did not block the binding of [18F]FOMPyD, which is in agreement with the data obtained in
rats.
To summarize the in vitro data, the tracer binding was most efficiently blocked with kinase
inhibitors based on the chemical scaffold of GW2580, such as FOMPyD and BOMPyD
followed in efficiency by selective type-II TKI PF-06273340. Type-I Trk inhibitor (R)-
IPMICF22 showed a lower and less statistically significant blocking efficiency, while CSF-1R
inhibitor BLZ945 did not block the binding of [18F]FOMPyD to wild-type rat and healthy
human brain tissues.
Baseline microPET scans revealed only moderate brain permeability of [18F]FOMPyD in wild-
type rats (whole brain SUVmax = 0.71 ± 0.04, n = 2) with the highest retention in the cortex
(Figure 6, A). The washout rate was relatively slow with more than half of the activity retained
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in the brain 90 minutes post-injection (SUV90 = 0.39 ± 0.01, n = 2). As a preliminary evaluation
of the binding specificity in vivo, we performed self-blocking experiments by spiking the tracer
dose prior to intravenous injection in a rat with the non-radioactive isotopologue. Indeed, the
radioactivity signal of [18F]FOMPyD in the brain was partially reduced by the administration
of non-radioactive FOMPyD, with the highest effect observed in the cortex at dosing
concentrations of 4–30 μg/kg. Blocking in the cortical regions (Figure 6, B) was consistently
low within the range of FOMPyD dosing concentrations at the putative equilibrium point 40
minutes past injection (ΔAUC40–90 = 11.1±0.7%; t(2) = 74.01, p < 0.001; n = 3), which suggests
limited binding specificity of [18F]FOMPyD to the target in vivo.
Conclusion. We developed an improved radiosynthesis of [18F]FOMPyD via copper-mediated
18F-fluorination of the boronic acid pinacolate precursor concomitant with the cleavage of four
Boc protecting groups in moderate radiochemical yield, high molar activity, radiochemical and
chemical purities, which allowed for its biological evaluation as a PET tracer for two unrelated
receptors implicated in neurodegenerative diseases. Autoradiography on rat and human brain
slices against a panel of several blocking agents revealed selective binding to TrkB/C rather
than CSF-1R, albeit with high non-specific binding. In baseline microPET experiments, the
tracer exhibits moderate brain uptake in wild-type rats, while the self-blocking scans revealed
mostly non-specific binding. Despite the shortcomings of [18F]FOMPyD as a PET tracer for
CNS imaging, the concerted methodology could be particularly useful for the 18F-fluorination
of acid-sensitive tracers via Cu-mediated displacement of boronic acid esters that require Boc-
protected precursors.
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Acknowledgments. This study was supported by a grant from the Alzheimer’s Society of
Canada 18-05 (for A. K.) and start-up grant from the Montreal Neurological Institute (for A.
K.) as well as Brain Canada Foundation with support from Health Canada (for McConnell
Brain Imaging Centre). Authors thank Drs. Gassan Massarweh and Jean-Paul Soucy, and
Messrs. Robert Hopewell, Dean Jolly and I-Huang Tsai for access to medical radioisotopes,
Ms. Karen Ross for assistance with microPET imaging and Messrs. Min Su Kang and Dhruv
Mehrotra for assistance with image data analysis.
Conflicts of interests. VBG is currently employed by the Bayer AG.
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Figure 1. Recently developed selective TrkB/C (A) and CSF-1R imaging probes (B) and
putative dual PET tracer [18F]FOMPyD (C).
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Figure 2. Synthesis of the precursor 6. Conditions: a) Cs2CO3, p-I-BnBr, s: MeCN, reflux; b)
3-morpholinopropionitrile, NaH, s: DMSO, 75°C; c) aniline·HCl, s: iPrOH, 40°C; d)
guanidine·HCl, NaOMe, s: EtOH, reflux; e) Boc2O, DMAP, s: THF; f) B2pin2, Pd(dppf)Cl2,
KOAc, s: DMSO, 80°C.
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Figure 3. Copper-mediated radiosynthesis of [18F]FOMPyD and formation of the side-product
of the protodeboronation reaction BOMPyD.
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Figure 4. In vitro characterization of [18F]FOMPyD on rat brain sections: sagittal sections (20
µm) were incubated with [18F]FOMPyD and either vehicle (DMSO) or 100 µM of non-
radiolabelled FOMPyD. A) Regional quantification for the in vitro autoradiography of
[18F]FOMPyD in normal rat brain. B) Representative in vitro autoradiography of
[18F]FOMPyD in the rat brain. Comparison between baseline and FOMPyD self-blocking. Ctx,
cortex; Cpu, caudate putamen; hipp, hippocampus; Th, thalamus; P, pons; SC, superior
colliculus. * and ** denote p values of <0.05 and <0.001, respectively.
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Figure 5. Characterization of [18F]FOMPyD binding blockade via in vitro autoradiography with
four different Trk or CSF-1R inhibitors (100 µM). A) Rat cortical regions (20 µm slices); B)
Human posterior cingulate cortex (PCC; 20 µm slices). * and ** denote p values of <0.05 and
<0.001, respectively. See Figure S4 and Figure S5 for source images, and Figure S6 for
inhibitor chemical structures.
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A
B
Figure 6. Time-activity curves of [18F]FOMPyD in wild-type rat brains. A) regional
distribution at the baseline scan (n = 2; see Figure S7 for source image). B) self-blocking with
FOMPyD reveals low uptake reduction in cortical regions (ΔAUC40–90 = 11.1±0.7%; t(2)
=
74.01, p < 0.001; n = 3) at dosing concentrations within 4–30 μg/kg range (blocking data is
shown in 1 rat).
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