Development of [18F]-labeled pyrazolo[4,3-e ]-1,2,4-triazolo[1, 5-c ]pyrimidine (SCH442416) analogs for the imaging of cerebral adenosine A 2A receptors with positron emission tomography

Article abstract of DOI:10.1021/jm500700y

Cerebral adenosine A_2A receptors (A_2ARs) are attractive therapeutic targets for the treatment of neurodegenerative and psychiatric disorders. We developed high affinity and selective compound 8 (SCH442416) analogs as in vivo probes for A_2ARs using PET. We observed the A_2AR-mediated accumulation of [¹⁸F] fluoropropyl ([¹⁸F]-10b) and [¹⁸F]fluoroethyl ([ ¹⁸F]-10a) derivatives of 8 in the brain. The striatum was clearly visualized in PET and in vitro autoradiography images of control animals and was no longer visible after pretreatment with the A_2AR subtype-selective antagonist KW6002. In vitro and in vivo metabolite analyses indicated the presence of hydrophilic (radio)metabolite(s), which are not expected to cross the blood-brain-barrier. [¹⁸F]-10b and [¹⁸F]-10a showed comparable striatum-to-cerebellum ratios (4.6 at 25 and 37 min post injection, respectively) and reversible binding in rat brains. We concluded that these compounds performed equally well, but their kinetics were slightly different. These molecules are potential tools for mapping cerebral A_2ARs with PET.

Full text of DOI:10.1021/jm500700y

Article
pubs.acs.org/jmc
Development of [18F]-Labeled Pyrazolo[4,3‑e]‑1,2,4-
triazolo[1,5‑c]pyrimidine (SCH442416) Analogs for the Imaging of
Cerebral Adenosine A2A Receptors with Positron Emission
Tomography
Shivashankar Khanapur,† Soumen Paul,† Anup Shah,‡ Suresh Vatakuti,§ Michel J. B. Koole,† Rolf Zijlma,†
Rudi A. J. O. Dierckx,† Gert Luurtsema,† Prabha Garg,‡ Aren van Waarde,† and Philip H. Elsinga*,†
†Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen,
Groningen, The Netherlands
‡Computer Centre, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali, India
§Department of Pharmacokinetics, Toxicology and Targeting, University of Groningen, Groningen, The Netherlands
ABSTRACT: Cerebral adenosine A2A receptors (A2ARs) are attractive therapeutic targets for the treatment of neurodegenerative
and psychiatric disorders. We developed high affinity and selective compound 8 (SCH442416) analogs as in vivo probes for
A2ARs using PET. We observed the A2AR-mediated accumulation of [18F]fluoropropyl ([18F]-10b) and [18F]fluoroethyl ([18F]-
10a) derivatives of 8 in the brain. The striatum was clearly visualized in PET and in vitro autoradiography images of control
animals and was no longer visible after pretreatment with the A2AR subtype-selective antagonist KW6002. In vitro and in vivo
metabolite analyses indicated the presence of hydrophilic (radio)metabolite(s), which are not expected to cross the blood-brain-
barrier. [18F]-10b and [18F]-10a showed comparable striatum-to- cerebellum ratios (4.6 at 25 and 37 min post injection,
respectively) and reversible binding in rat brains. We concluded that these compounds performed equally well, but their kinetics
were slightly different. These molecules are potential tools for mapping cerebral A2ARs with PET.
(cAMP).4 The subtypes differ in size (A1, A2A, A2B, and A3
INTRODUCTION
■
consist of 326, 409, 328, and 318 amino acids, respectively) and
Adenosine, an endogenous signaling substance, is a purine
exhibit unique tissue distributions.5
ribonucleoside that is composed of adenine (purine base) and
ribose (sugar molecule).1 It functions as a cytoprotectant and
Over the last 30 years, the most extensively studied AR
subtypes are the biochemically and pharmacologically well-
characterized high-affinity A1Rs and A2ARs. Adenosine activates
these receptors at nanomolar concentrations.2 A1Rs are widely
distributed in the human brain, with the highest densities being
found in the hippocampus, cerebral cortex, thalamic nuclei, and
dorsal horn of spinal cord, whereas A2ARs are highly expressed
in the dopamine-rich regions of the brain, and the highest levels
of A2AR expression occur in the striatum (caudate-putamen,
nucleus accumbens, and olfactory tubercle), globus pallidus and
substantia nigra.6−9 Lower levels of A2ARs occur in the
neuromodulator in response to organ and tissue stresses under
both physiological and pathological conditions.2 In the brain,
adenosine plays an important role in the regulation of both
neuronal and glial cell functions. Furthermore, it counteracts
glutamate excitotoxicity and cytokine-induced apoptosis.3 Its
actions are mediated through the activation of four subtypes of
G-protein coupled adenosine receptors (ARs), namely A1, A2A,
2
A2B, and A3.
Adenosine A1 receptors (A1Rs) and adenosine A3 receptors
(A3Rs) are G-protein-coupled binding sites for adenosine that
inhibit adenylyl cyclase, whereas adenosine A2A receptors
(A2ARs) and adenosine A2B receptors (A2BRs) stimulate
adenylyl cyclase via GS proteins and promote the formation
of the second messenger cyclic adenosine monophosphate
Received: May 23, 2014
Published: July 25, 2014
© 2014 American Chemical Society
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Figure 1. Structures of some selected AR agonists, an antagonist KW6002, and A2AR PET imaging agents.
hippocampus, cerebral cortex, amygdala, cerebellum, brainstem,
and hypothalamus.10−13
PD24 or AD.25 Along with D2Rs, A2AR antagonists attenuate the
overactivity of the indirect dopamine pathway observed during
PD, restore balance between the direct and indirect output
pathways, and suppress the neurodegenerative process by
modulating the activity of cortico-striato-pallido-thalamocort-
ical (CSPTC) pathways.20,26,27
The activation of AlRs (e.g., by A1R agonists) increases sleep,
inhibits seizures, reduces anxiety, and promotes neuro-
protection. On the other hand, A1R antagonists are anxiolytics,
are beneficial in the treatment of cognitive disorders, cardiac
arrhythmia, asthma, and other respiratory disorders, and are
therapeutic drugs for kidney protection.14 A2AR agonists are
implicated in tissue repair, which involves a series of
coordinated and overlapping phases such as inflammation,
wound healing, angiogenesis, and tissue reorganization.15−17
The vasodilating effect of A2AR agonists (adenosine and
regadenoson) has been fully validated (see Figure 1).
Adenosine (Figure 1, compound 1) is used for the treatment
of paroxysmal supraventricular tachycardia,3 whereas regade-
noson (Figure 1, compound 2) is currently marketed for use as
a pharmacologic stress agent in (radionuclide) myocardial
perfusion imaging.18
A2ARs mediate potential neuroprotective and neurotoxic
effects in addition to modulating dopaminergic neurotransmis-
sion in the basal ganglia through the antagonistic interactions
between A2ARs and dopamine D2 receptors (D2Rs).19−21
However, their role in neurodegenerative diseases such as
Parkinson’s disease (PD) and Alzheimer’s disease (AD) is
highly controversial.21 Nevertheless, based on preclinical
studies, A2AR antagonists have potential benefits in the
treatment of neurodegenerative and psychiatric disorders such
as PD, AD, neuroinflammation, ischemia, spinal cord injury,
drug addiction, and other conditions.18,20−23 Moreover, recent
epidemiological studies have established that the regular
consumption of caffeine (a xanthine derivative and AR
antagonist) is associated with a lower risk for developing
Positron emission tomography (PET) can noninvasively
assess the functional status of CSPTC pathways in PD and
related disorders.28 High-affinity antagonistic radioligands that
are selective for the A2ARs can be used to assess changes in
A2AR density during disease progression and to monitor the
effects of therapy on these changes.2 Moreover, they can be
employed to assess the occupancy of the receptor population
by therapeutic drugs in the human brain, which will allow the
correlation of receptor occupancy with dose, drug/tracer
plasma levels, and therapeutic effects.2,18,29,30 We focused on
A2AR antagonist core structures as PET ligands (instead of
A2AR agonist structures) because agonist PET tracers may only
bind to the high-affinity state of the receptors, resulting in a
poor signal-to-noise ratio.31 Moreover, the in vivo vulnerability
to competition by endogenous adenosine may hamper the
quantification of the total number of binding sites.31
The design and development of novel A2AR antagonist PET
ligands is a key research topic because current xanthine-based
tracers suffer from several disadvantages, including high
nonspecific binding, low signal-to-noise ratios, and therefore,
target sites in the brain are barely visible. Furthermore, the use
of xanthine-based tracers suffer from a photoisomerization
problem, low selectivity toward A2AR.2,29,32−38 On the basis of
these considerations, nonxanthine compounds were developed
and evaluated in many preclinical and clinical studies for the
assessment of cerebral A2ARs.18
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A high degree of selectivity and appropriate combinations of
lipophilicity, molecular weight, and affinity are important in the
development of ideal in vivo A2AR PET brain tracers.39 For a
compound to cross the blood-brain-barrier (BBB), a relatively
small molecular weight (400 to 500 Da) and moderate
lipophilicity (approximate range of log P is 2 to 3.5) are
optimal.39−41 High lipophilicity causes unacceptable binding to
plasma proteins and lipid bilayers, resulting in high levels of
nonspecific binding in the brain.39 Low lipophilicity decreases
the penetration of PET agents across the BBB. In addition, the
tracer’s affinity must balance the opposing goals of tight binding
and significant washout from the brain. Furthermore, the easy
and rapid (within 3 half-lives) incorporation of radionuclides
into the appropriate precursor molecules is necessary. Finally,
the formation of lipophilic radioactive metabolites should be
negligible because the presence of resulting radiometabolites in
the target tissue would impede the quantification of the PET
data using kinetic models.39,42
Table 1. Docking Analysis of A2A Antagonists Using GOLD
Software
Rodent studies with nonxanthine tracer 7-(3-(4-[11C]-
methoxyphenyl)propyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidine-5-amine [11C]-8 ([11C]SCH442416;
Figure 1)43,44 and its 2-[18F]fluoroethyl derivative [18F]-10a
([18F]MRS5425 = [18F]FESCH in Scheme 3)47,48 suggest their
potential value for in vivo mapping of A2ARs.43−48 However,
high nonspecific binding and a lower striatum (target)-to-
cerebellum (nontarget) ratios (4.6 for [11C]-8 at 15 min; no
target-to-nontarget data available for [18F]-10a) than [7-
methyl-11C](E)-8-(2,3-dimethyl-4-methoxystyryl)-1,3,7-trime-
thylxanthine [11C]-3 ([11C]KF21213; a xanthine-based tracer,
10.5 at 60 min, Figure 1)38 were associated with these tracers,
and these compounds will require further evaluation in human
subjects.44,48 Furthermore, [11C]-3 (a tight binding tracer
suffers from several limitations such as photoisomerization, an
impaired kinetic profile, low BBB penetration, and poor water
solubility.2,44
All of the A2AR antagonist PET ligands that have been
successfully evaluated in humans to date are [11C]-labeled. The
radioisotope 18F has advantages of higher specific activity and a
longer physical half-life (109.8 min vs 20.4 min) than [11C]
ligands; these advantages allow the tracer to be distributed to
remote imaging centers without cyclotron facilities and to
achieve longer biodistribution and scanning times for a better
assessment of the dissociation rate constant (Koff).48,49 On the
basis of the above considerations, favorable docking results and
because compound 8 demonstrated an appropriate lipophilicity
for CNS imaging in addition to the highest A2AR affinity and
subtype-selectivity, the present study utilized 8 as a lead
compound to develop a novel radiofluorinated A2AR ligand. We
designed and prepared an 7-(3-(4-(3-[18F]¯uoropropoxy)-
phenyl)propyl)-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-amine derivative [18F]-10b
([18F]FPSCH in Scheme 3) and compared its pharmacoki-
netics and biodistribution in healthy rats with those of [18F]-
10a and [11C]-8 to optimize the length of the fluoroalkyl chain,
which could affect both A2AR affinity and selectivity.
derivatives and A2ARs. The major ligand binding interactions
are both polar and hydrophobic in nature and occur with
residues in trans-membrane domains 3, 5, 6, and 7. Residues
from the second extracellular loop (ECL2) outline the upper
part of the binding cavity (Figure 2B). In our study, 7-(3-(4-(2-
fluoroethoxy)phenyl)propyl)-2-(furan-2-yl)-7H-pyrazolo[4,3-
e][1,2,4]triazolo [1,5-c]pyrimidin-5-amine 10a (FESCH,
Scheme 2) and 7-(3-(4-(3-fluoropropoxy)phenyl)propyl)-2-
(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-
5-amine 10b (FPSCH, Scheme 2) had binding modes that were
similar to the cocrystallized 4-(2-[7-amino-2-(2-furyl)[1,2,4]-
triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol 17
(ZM241385, fifth compound in Table 1) conformation (Figure
2A), including the important hydrogen bond interactions with
RESULTS
■
Molecular Docking. Table 1 shows the GOLD fitness
scores and important interactions for the docked ligands. A
molecular docking study was performed to elucidate the
intermolecular interactions between the 7-(3-(4-
methoxyphenyl)propyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidine-5-amine 8 (SCH442416, Scheme 2)
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Figure 2. (A) Interaction between antagonist 17 and A2AR binding site residues. (B) Docked conformation of 10b with A2AR, viewed from the
membrane and extracellular (ECL) sides, showing ECL2-folded into the binding cleft. TMs 1−7 are labeled for reference. (C and D) Close-up view
showing the pose for (C) 10a and (D) 10b in the A2AR binding site residues. The residues and compounds are shown in stick models. The residues
involved in ligand binding are labeled and represented as violet sticks, oxygen atoms are shown in red, nitrogen atoms are shown in navy blue, and
hydrogen atoms are shown in gray. The compounds are represented as purple sticks, the fluorine atom is represented in cyan, and the rest of the
indicated atoms are similar to A2AR residues. The dashed lines in black indicate hydrogen bonds.
the active site residue Asn253 and a π−π stacking interaction
with Phe168 of A2AR. The exocyclic free amino group of the
pyrimidine ring structure of the tricyclic core and the oxygen of
the furan ring make strong H-bond interactions with Asn253 of
the receptor. This finding is consistent with the results from
site-directed mutagenesis studies of the A2AR, which suggest
that this amino acid is critical for ligand binding.50 Moreover,
the π−π stacking interaction between Phe168 and His250
stabilizes the binding pose of the compound within the active
site. Compound 8 derivatives, including the A2AR-bound crystal
structure, 17, are oriented perpendicular to the plane of the cell
membrane, with their flexible hydrocarbon side chain located in
the extracellular domain. An analysis of ligand-bound crystal
structure and literature evidence47,51 suggests that ECL2 helps
in ligand binding at the A2AR. We explored the impact of
structural variability at the terminal phenolic position of 8 in
the GOLD docking scores. As reported previously,47,51
conformational flexibility was also noted in our experiment;
the terminal phenolic side chain forms a polar interaction with a
crystallographic water molecule at the extracellular matrix of
A2AR. As expected, N6-cyclopentyl-N8-isopropyl-N8,9-dimethyl-
9H-purine-6,8-diamine 18 (LUF5608, sixth compound in Table
1), a high affinity A1R antagonist and negative-control, yielded a
very low docking score due to a lack of hydrogen bond
formation with the active site residues of the receptor, further
authenticating the findings of the docking study and indirectly
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a
Scheme 1. Synthesis of Fluoroalkyltosylates
a
Reagents and conditions: (i) tosyl chloride, pyridine, DCM, room temperature (RT), stirring 3 h; (ii) tetrabutyl ammonium fluoride trihydrate,
acetonitrile, overnight reflux.
a
Scheme 2. Disconnection Retrosynthetic Scheme for the Reference Fluoroalkylated Compound 8 Analogs (10a and 10b)
a
Reagents and conditions: (iii) BBr3, CH2Cl2, RT, 2 h; (iv) 5, Cs2CO3, MeOH, reflux, 1 h; and (v) 7, Cs2CO3, MeOH, reflux, 16 h.
confirming the specificity of 10b and 10a toward A2AR over
A1R.
[18F]fluoroalkylation of the phenol precursor 9 (Scheme 3).
Table 2 lists the decay-corrected radiochemical yields, specific
radioactivities, calculated partition coefficient (clogP) and
experimentally determined distribution coefficient (LogD7.4)
values for [18F]-10a and [18F]-10b. For both tracers, the
radiochemical purity was >98%, and the total synthesis time,
Chemical and Radiochemical Synthesis. The demethy-
lation of commercially available 8 using boron tribromide
(BBr3) resulted in a quantitative yield of 4-(3-(5-amino-2-
(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-
7-yl)propyl)phenol, 9 (precursor of compound 8, Scheme 2).47
A retrosynthetic approach was adopted for the synthesis of
reference standards (10a and 10b, Scheme 2), which were
prepared by reacting the phenol precursor (9) with the
appropriate fluoroalkyltosylate (5 or 7 in Scheme 1, selective
fluoroalkyaltion) in 35% and 25% yields, respectively. It was
adopted for the synthesis of reference standards (10a and 10b,
Scheme 2). The two radiolabeled analogs, [18F]-10a and [18F]-
10b, were synthesized by a two-step two-pot procedure starting
with the corresponding [18F]fluoroalkyl synthon ([18F]-5 or
[18F]-7) made from [18F]fluoride and the appropriate
ditosylate precursor (6 or 11), followed by the selective
including quality control, was 114
5 min (n = 18). The
identities of the tracers were confirmed by spiking with
authentic cold compounds in reversed-phase HPLC (RP-
HPLC).
Ligand Metabolism. In Silico Metabolite Analysis. The
predicted sites of metabolism are highlighted for parent
compound 10b in Scheme 4 (data not shown for 10a and
8). The possible metabolic routes can be ranked in the
following order: C-hydroxylation > N-oxidation > O-deal-
kylation.
Human Liver Microsomal Metabolite Analysis. Table 3
summarizes the modifications that were detected with an
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a
Scheme 3. Radiosynthesis of [18F]-10b and [18F]-10a
a
Reagents and conditions: (vi) K[18F]F−K2.2.2−K2CO3, acetonitrile, 6, 1,2-ethanediol di-p-tosylate, 125 °C, 10 min; (vii) tetrabutyl ammonium
hydroxide (40% aq.), acetonitrile, sealed conditions, 115 °C, 15 min.
Table 2. Radiosynthesis and Lipophilicity Data
clogP
a
b
radioligand
radiochemical yield and purity (%)
specific radioactivity (GBq /μmol)
22.5
136 13
Chemsketch 12.01
2.98 0.98
Chembiodraw ultra 12.0
LogD7.4
[18F]-10a
[18F]-10b
7
8
2 and ≥98
2 and ≥98
5
3.18
3.41
3.16 0.03
3.41 0.11
3.27 0.98
b
a
Overall radiochemical yields based on starting wet [18F]fluoride and corrected for decay. Experimental value.
In Vitro Ligand Stability Test. The in vitro stability of the
two [18F]tracers ([18F]-10a and [18F]-10b, Scheme 3) in
different solutions such as PBS, saline, rat plasma, and human
plasma was determined at 37 °C. After 1 and 2 h of incubation,
radio-TLC analysis showed that 95−97% of both tracers were
still intact, except in the saline solution. In the saline solution,
multiple spots were observed as detected by radio-TLC for
both tracers, and the radioactivity corresponding to the intact
tracers was only 85−90%.
Scheme 4. Sites of Metabolism Predicted for 10b by
SMARTCyp Webservice
a
In Vitro Autoradiographic Experiments. Figure 4 shows
autoradiographic images of frozen rat brain sections that were
incubated with [18F]-10a and [18F]-10b. In control sections, a
clear difference was noted between the receptor-rich striatum
and the receptor-poor cerebellum. The mean striatum-to-
cerebellum ratios were 2.75 0.12 ([18F]-10a) and 2.99 0.16
([18F]-10b). In the presence of an excess (2 μM) of the A2AR-
specific antagonist 8-[(1E)-2-(2-(3,4-dimethoxyphenyl)-
ethenyl]-1,3-diethyl-3,7-dihydro-7-methyl-1H-purine-2,6-dione,
12 (KW6002 in Figure 1), the binding of the tracer to the
striatum was strongly reduced and the striatum-to-cerebellum
ratio decreased to unity (n = 3). Specific binding, as assessed by
a blocking study, was 57−62% and 64−67% of the total uptake
in the striatum of [18F]-10a and [18F]-10b, respectively.
Micro PET Images. PET images acquired after injection of
[18F]-10a and [18F]-10b are presented in Figure 5. The two
radioligands displayed similar regional distributions that
corresponded to the known regional A2AR densities in the rat
brain.7−9,11 In order to prove specific binding, we have used
vehicle-control and blocker animals (Please refer in vivo and in
vitro selectivity of the experimental section for more details). In
a
The same predicted metabolic sites are also applicable to compounds
8 and 10a.
ultrahigh-performance liquid chromatography/quadrupole-
time-of-flight-mass spectrometer (UHPLC/Q-ToF-MS) and
analyzed by Metabolynx after incubating compound 8 with
human liver microsomes. The relative production of the
different metabolites over time is presented in Figure 3. None
of these modifications and major demethylated metabolites
were detected in negative control and positive control
(verapamil) incubations, respectively, with human liver micro-
somes. Under these conditions, the m/z ratio increased by 16,
which is most likely the result of N-oxidation or C-
hydroxylation. The m/z ratio also decreased by 14, which
may be due to a demethylation reaction. Moreover, a fluorine-
containing metabolite of 10b and free fluoride was observed,
indicating defluorination, similar to previously reported results
for 10a.47
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Table 3. Human Liver Microsomal Metabolite Analysis
cerebral uptake of the tracers was strongly reduced, and
regional differences in tracer uptake were no longer observed.
In Vivo Radioligand Kinetics and Metabolism. Kinetics
of Radioactivity in Brain. The cerebral kinetics of radioactivity
after the injection of [18F]-10a and [18F]-10b are presented in
Figure 6 (panels A−D). In vehicle-treated control animals (n =
6), the uptake of radioactivity rapidly increased to a maximum
(2.5 min after injection), which was followed by an exponential
washout (panels A and C). In animals treated with 12 (n = 6),
the cerebral uptake of 18F was strongly reduced, and the
radioactivity was rapidly washed out from all brain regions
(panels B and D). The difference in the striatal uptake of 18F in
control and pretreated rats was statistically significant at most
time points.
We estimate receptor occupancy on the basis of PET-
standardized uptake values (PET-SUVs) (at the time of
maximum uptake), reported A2AR densities (953 fmol/mg
protein in rat striatum52) and injected tracer doses in
nanomolar amounts. Assuming that brain tissue contains 10%
protein, we calculated that less than 2% and 8% of the cerebral
A2AR population was occupied by [18F]-10b and [18F]-10a,
respectively, in both control- and blocker-treated rats.
Figure 3. Relative amounts of several modifications over 90 min
during incubation of (8) with human liver microsomes. The error bars
indicate the standard deviation.
vehicle-control animals, the striatum was clearly visualized. The
extrastriatal binding of both tracers was hardly visible, but
strong uptake was observed in the skull bone. When animals
were pretreated with the A2AR antagonist 12 (1 mg/kg), the
Radioactivity Kinetics in Plasma. A rapid, biexponential
plasma clearance was observed in all groups. Pretreatment did
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Figure 4. Autoradiographic images of sagittal sections of rat brains after 90 min of incubation with (A and B) [18F]-10b or (C and D) [18F]-10a in
the (B and D) presence or (A and C) absence of an excess of a known A2AR-selective antagonist, 12 (2 μM).
not significantly affect the clearance of radioactivity from the
plasma compartment.
DISCUSSION
■
We have evaluated [18F]-10a and [18F]-10b as PET tracers for
the cerebral imaging of A2ARs; these tracers may provide many
logistic advantages and can be used in centers without an on-
site cyclotron. Thus, we synthesized fluorinated molecules
based on a pyrazolo-triazolo-pyrimidine template (compound
8) that is known to cross the BBB because of its appropriate
lipophilicity (clogP = 2.9), molecular weight, charge, and
hydrogen bonding.39,43,44 The molecular docking approach
provides valuable atomic-level insight into the behavior of a
small molecule in the binding site of the protein. It also
provided insight into the binding mode of compound 8
derivatives to the active site of the receptor. Compound 8 and
its fluoro analogs (10a and 10b) had better GOLD fitness
scores than the clinically studied PET tracer (E)-8-(3,4,5-
trimethoxystyryl)-1,3-dimethyl-7-[11C]methylxanthine ([11C]-
KF18446) and the A2AR-bound crystal structure 17. GOLD
scores are good indicators to predict the binding orientations of
compound 8 derivatives. A higher score predicts better binding
orientations with the receptor residues. We have used the A1R
decoy, 18-as negative control, to validate the quality of the
GOLD scoring function.53 Small structural changes of the
phenoxy substituent appeared to be well-tolerated, and this is
substantiated by our in vivo study. Subtype selectivity can be
expected because the lead compound 8 shows a >10,000-fold
selectivity for the A2AR subtype compared to other AR
subtypes. Moreover, the binding affinities of 8 (Ki = 0.5 nM)
and 10a (Ki = 12.4 nM) to the A2AR were adequate for
imaging.43,47 Even though the reported Ki value of 53.6 nM for
10b54 is much lower than that of the lead compound 8, the
affinity data predict faster clearance than 8 and 10a and more
preferable brain kinetics for the quantitative evaluation of the
In Vivo Metabolite Analysis. An unidentified radio-
metabolite with a Rf value of 0−0.1 was observed in rat plasma
(the Rf value of authentic [18F]-10b was 0.6). The fraction of
total plasma radioactivity representing the parent compound
decreased to 66 16% at 60 min and 53 20% at 90 min.
Pretreatment with 12 did not affect the rate of tracer
metabolism. The fraction of total plasma radioactivity
representing [18F]-10a was 46
17% at 60 min and 36
14% at 90 min.
Ex Vivo Biodistribution Data. The biodistribution data for
both tracers are shown in Figure 7. After pretreatment with 12,
the uptake of both compounds was reduced in the A2AR-rich
striatum (approximately 69% of [18F]-10b and 45% for [18F]-
10a). For [18F]-10b, the effect of the blocker was statistically
significant in the frontal cortex and striatum, whereas for [18F]-
10a (n = 3), no significant effect of the blocker was observed in
any of the brain regions; however, the greatest decrease of the
tracer uptake was observed in the striatum. Striatum-to-
cerebellum ratios can be used as indices for the in vivo binding
of the tracers to A2ARs. Striatum-to-cerebellum ratios of 3.5 and
2.1 were reached at 106 min postinjection for [18F]-10b (n = 6)
and [18F]-10a (n = 3), respectively. The ratios of the uptake in
other regions to the cerebellum were approximately equal to
one. The standard uptake values in the skull bone (2.06 0.58)
for [18F]-10b were significantly higher than those of [18F]-10a
(0.29 0.05).
For both tracers, the plasma-to-blood ratio was greater than
one, and the negligible binding to red blood cells in pretreated
and control animals indicated that the radioligands preferen-
tially distributed to the plasma.
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Figure 5. Small-animal PET images of a coronal plane of rat brains after injections of (A and B) [18F]-10a or (C and D) [18F]-10b. The images
represent the summed frames from 17 to 90 min post injection. (A) Vehicle-control (left); (B) a compound 12-treated animal ([18F]-10a) (right).
(C) Vehicle-control (left); (D) a compound 12-treated animal ([18F]-10b) (right). The images were normalized for body weight and injected dose.
ligand−receptor binding. The pyrazolo-triazolo-pyrimidine
scaffold allows for the easy and quick incorporation of an
[18F] label in the acidic phenol group, and this phenoxy
substituent can also be used to modify the lipophilicity of the
compound. Fluoroalkyl chain lengthening beyond the
fluoropropyl substituent results in a higher molecular weight
(MW) and lipophilicity for a compound. It has been suggested
that the MW should be kept below 450 Da to facilitate brain
penetration with fewer side effects such as high rapid metabolic
turnover, poor absorption, and toxicity.55 High lipophilicity
causes unacceptable binding to plasma proteins, decreasing the
free drug concentration available to pass the BBB, or binding to
hydrophobic protein targets other than the desired one,
resulting in high levels of nonspecific binding in the
brain.39,55 On the basis of these considerations, 10b was
selected as a novel candidate for radiolabeling to obtain the
expected lipophilicity and a MW that ensures the crossing of
the BBB.
experiment and the predictions of a two-dimensional (2D)
method (SMARTCyp) describing CYP450-mediated drug
metabolism.47,56 In contrast to the results obtained from a
SMARTCyp prediction of drug metabolism (please refer to
Ligand Metabolism module in the Results section), the
metabolic routes of compound 8 after incubation with liver
microsomes can be ranked in the following order: O-
dealkylation > parent-C10H12O > N-oxidation or C-hydrox-
ylation > deamination (Figure 3, Table 3). A stability test
indicated that both 10a and 10b are highly stable in vitro.
Because of the observed multiple spots as detected with radio-
TLC in a saline solution, we used PBS instead of saline in the
formulation of the tracers. The fraction of total plasma
radioactivity representing [18F]-10b was 18−20% higher than
that of [18F]-10a at both 60 and 90 min in our in vivo
metabolite analysis. However, stronger skull bone radioactivity
uptake (i.e., stronger defluorination) was observed with [18F]-
10b than [18F]-10a in both an ex vivo biodistribution study and
a small-animal PET (microPET) image analysis. In our imaging
studies (especially with [18F]-10b), the accurate quantitation of
the radioactivity in the frontal cortex was difficult due to
spillover from radiofluorine in the skull bone.
Computational prediction of the sites of cytochrome P450
(CYP450)-mediated metabolism and an in vivo plasma radio-
TLC metabolite analysis indicated the formation of polar
metabolites, which are not expected to cross the BBB. The
results obtained after incubation of 10b with hepatic micro-
somes were in good agreement with a previously reported
A retrosynthetic approach for 10a and 10b synthesis was
successfully applied to avoid a cumbersome and time-
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Figure 6. (A−D) Kinetics of (A and B) [18F]-10a and (C and D) [18F]-10b-derived radioactivity in the rat brain. The error bars indicate the SEM.
●
▼
(A and C) Vehicle-control animals (left); (B and D) compound 12-treated animals (right). , striatum; , cerebellum.
consuming scheme involving 8 reaction steps. In the synthesis
of compound 10b, 3-fluoropropyl tosylate 7 yielded a slightly
better result than 1-bromo-3-fluoropropane (25% vs 19% yield)
because tosylate is a better leaving group than bromide. Tracers
were successfully synthesized using a two-pot two-step
procedure (Scheme 3). The [18F]fluoroalkylation of phenol
precursor 9 using corresponding intermediate fluorosynthons
([18F]-5 or [18F]-7) yielded the desired ligands [18F]-10a and
[18F]-10b at moderate yields (7−8%) and satisfactory specific
activities (Table 2). The average radiochemical yield of the
Our radiosynthetic procedure for [18F]-10a is much faster (by
15−20 min) than the existing procedure.47 The applied
radiolabeling approach is versatile; we can quickly adopt the
same procedure for the radiosynthesis of both compounds
[18F]-10a and [18F]-10b. The radiochemical purities were also
adequate and amounted to more than 98% of the total
radioactivity as determined by UHPLC quality control.
In vitro autoradiography (ARG) confirmed the selectivity of
[18F]-10a and [18F]-10b for A2A Rs. The tracer binding pattern,
especially in the striatum and other parts of the brain, was
comparable with ex vivo biodistribution readings. The regional
distribution of radioactivity in the rat brain after the injection of
[18F]-10a and [18F]-10b also suggests that these tracers are
capable of measuring regional A2AR densities. After pretreat-
ment with a subtype-selective xanthine antagonist, 12, the
tracer uptake in the striatum was greatly suppressed, and
regional differences were no longer present. In the biodis-
tribution and PET studies, the uptake of [18F]-10b in the
cerebellum and frontal cortex (areas lacking A2ARs) was also
decreased after pretreatment with 12. The most logical
explanation for the specific binding in the cerebellum is that
[18F]-5 or [18F]-7 obtained was 50
fluoroalkylation conversion was approximately 25
5%, whereas the final
5%.
Purification by HPLC and solid phase extraction provided a
decay-corrected radiochemical yield of 7−8%. The long
evaporation step of the captured eluate [14 mL of hexane/
ether (3:1)] during the purification of the [18F]-5 or [18F]-7
and the losses that occurred during the other manipulations of
the synthesis accounted for the moderate radiochemical yields
of the synthesized tracers [18F]-10a and [18F]-10b. The
purification of the [18F]-5 or [18F]-7 by RP-HPLC and C-18
light Sep-Pak columns may improve the radiochemical yield.
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Figure 7. Cerebral biodistribution data of [18F]-10b and [18F]-10a at 106 min after injection. The error bars indicate the SEM. BOLF = Bulbus
olfactorius, CERE = Cerebellum, FCor = Frontal cortex, Stria = Striatum, Hipp = Hippocampus, Medu = Medulla, PTOC = Parietal/Temporal/
Occipital Cortex.
Figure 8. Striatum-to-cerebellum ratios of [18F]-10b and [18F]-10a as a function of time. The solid and broken lines represent [18F]-10b and [18F]-
10a, respectively. The error bars indicate the SEM.
the endothelium and blood vessels express A2ARs, even if the
brain tissue does not.18,57 Taking into account the findings that
pretreatment with 12 reduces the distribution volume of
candidate reference tissues such as the cerebellum, we choose
not to quantify the blocking effect using a (simplified) reference
tissue model, 2-tissue compartment model, and Logan analysis.
It is evident from the microPET images that the extrastriatal
binding of the [18F]-tracers was barely visible, except for the
strong uptake in skull bone due to defluorination, whereas in
the case of xanthine PET ligands, the extrastriatal retention of
radioactivity was visualized in the cortex, cerebellum, and
thalamus.34 Because of estimated receptor binding of the
injected tracer mass (<2%−8%), we do not expect any
pharmacological effects or change in the physiological states
of the A2AR system during our microPET study.
The striatal uptake of both [18F]-ligands was clearly
visualized using PET scans; both tracers reached a striatum-
to-cerebellum ratio of approximately 4.6, which is similar to the
experiments with [11C]-8 result (4.6 0.27).44 However, the
maximum ratio for [18F]-10b was reached at a later time point
(37 min) than that of [18F]-10a (25 min) and [11C]-8 (15
min), most likely because of the higher lipophilicity of [18F]-
10b (Figure 8). Lipophilicity may prolong the circulating half-
life of a tracer, resulting in extended availability for binding to
A2ARs. After the maximum had been reached, the concentration
of [18F]-10a in the brain remained fairly stable until 30 min
after injection (similar to [11C]-8), whereas the concentration
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of [18F]-10b showed a somewhat stronger washout. The
cerebral kinetics of both radioligands were compatible with the
duration of a PET scan (Figure 8).
found to be ≥95%. A sample solution (100% acetonitrile) with a
concentration of approximately 0.1 mg/mL was prepared, and an
injection volume of 5 μL was used. Data were acquired as described in
the in vitro microsomal metabolite analysis section using a 4 min
gradient starting from 0.1% formic acid in 95% water for 1.5 min, next
with an 80% acetonitrile solution for 1 min, and finally with 0.1%
formic acid in 95% water at a flow rate of 0.6 mL/min.
A disconnection retrosynthetic strategy was designed for the easy
preparation of the 10a and 10b analogues.47,54 Compound 8, in
dichloromethane (DCM) was demethylated using BBr3; this yielded
the precursor 9. This precursor was used without further purification
for both the chemical and radiochemical syntheses.
Precursor 9 was characterized by ESI-HRMS m/z 376.1522 [M +
H]+, C19H17N7O2.H+: Calcd 376.1522; 1H NMR (400 MHz,
Methanol-d4): δ 8.08 (s, 1H), 7.78 (dd, J = 1.7, 0.7 Hz, 1H), 7.30
(dd, J = 3.5, 0.7 Hz, 1H), 6.92−6.83 (m, 2H), 6.66 (dd, J = 3.6, 1.8 Hz,
1H), 6.60−6.50 (m, 2H), 4.28 (t, J = 6.9 Hz, 2H), 2.47 (t, J = 7.5 Hz,
2H), 2.26−1.96 (m, 2H).
CONCLUSION
■
[18F]-10b and [18F]-10a could be prepared using a two-step
procedure. Both radioligands showed a distribution in the rat
brain, corresponding to the regional A2AR densities known
from in vitro ARG and binding assays. Experimental LogD7.4
values, plasma metabolite analysis and microPET data analysis
results suggest that these radiopharmaceuticals are potentially
useful for mapping cerebral A2ARs. The molecular docking
studies, similar target-to-nontarget ratios in ex vivo brain
biodistribution and kinetic analysis, distribution patterns and
only slightly different kinetics suggest that small increases in the
length of the fluoroalkyl chain do not result in impaired
pharmacokinetics and metabolic stability.
2-[19F]-Fluoroethyl Tosylate (5). 2-Fluoroethanol 4 (1.28 g, 20
mmol) was dissolved in 20 mL of DCM at 0 °C. Tosyl chloride (4.19
g, 22 mmol) and pyridine (1.74 g, 22 mmol) were added to this
solution. The reaction mixture was then allowed to warm to room
temperature and stirred continuously for 3 h. After the addition of
water (100 mL) and saturated ammonium chloride solution (50 mL),
the mixture was extracted with DCM (3 × 150 mL). The combined
organic fractions were washed thoroughly with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo to generate the
crude product. The crude product was then purified by silica column
purification using n-hexane:DCM (50% v/v) as an eluent to generate
pure 5 (Scheme 1) in the form of colorless oil, Rf 0.34. The isolated
yield was 51%.
EXPERIMENTAL SECTION
■
In silico Docking Studies. The coordinates of the X-ray crystal
structure of human A2AR in complex with the triazolotriazine 17, a
high-affinity and selective A2A antagonist (Figure 2, protein data bank
ID: 3PWH, www.rcsb.org/pdb), plus those of some well-known A2AR
antagonists, were used as a basis for ligand docking to investigate the
binding properties of 10a and 10b (Figure 2, panel B and C). The
three-dimensional (3D) structures of these molecules were drawn
using the SYBYL 7.1 molecular modeling package.58 Next, the
constructed molecules were energy minimized using the Powell
method for 10000 iterations with 0.05 kcal/mol as a gradient.59
A
ESI-HRMS m/z 241.0309 [M + Na]+, C9H11O3FS.Na+: calcd
1
tripos force field and Gasteiger-Huckel charges were used during
̈
241.0310; H NMR (400 MHz, CDCl3): δ 7.80 (d, J = 8.3 Hz, 2H),
minimization.60 The GOLD 5.1 docking protocol was employed for
the analysis.61 Hydrogens were added to the crystal structure, and a
grid of size 10 Å was constructed around the cocrystallized ligand. The
cocrystallized ligand was removed after the preparation of the grid.
The docking was then carried out to predict the binding mode of the
same cocrystallized ligand, and a comparison was made with the
crystallographically observed positions (n = 3). Compound 18, a high
affinity A1R antagonist was included as a decoy compound in a pool of
known A2AR antagonists to further validate the docking protocol. The
GOLD fitness score and the various interactions, such as hydrogen
bonding/π−π interactions with important active site residues from the
validated docking protocol, were selected as evaluation criteria. The
docking poses were analyzed using Pymol (Delano Scientific LLC, San
Carlos, CA).
Materials (Chemical Synthesis). The compounds 8 and 12 were
purchased from Axon Medchem BV (Groningen, The Netherlands).
1,3-Propanediol di-p-tosylate (6) and 1,2-ethanediol di-p-tosylate (11)
were procured from Aldrich (Sigma-Aldrich, The Netherlands).
Tetrabutylammonium hydroxide solution (40% in H2O) was
purchased from Fluka (Sigma-Aldrich, The Netherlands). All other
chemicals were of analytical grade and obtained from commercial
suppliers such as Rathburn, Sigma, Merck, and others. These chemicals
were used in the syntheses without any further purification. Oxygen-
or moisture-sensitive reactions were carried out under an atmosphere
of dry N2 or argon using dried glassware.
7.35 (d, J = 8.3 Hz, 2H), 4.62 (dd, J = 5.0, 3.2 Hz, 1H), 4.50 (dd, J =
5.0, 3.2 Hz, 1H), 4.29 (dd, J = 4.9, 3.3 Hz, 1H), 4.22 (dd, J = 4.9, 3.3
Hz, 1H), 2.44 (s, 3H).
3-[19F]-Fluoropropyl Tosylate (7). Tetrabutyl ammonium fluoride
trihydrate (52.8 mg, 0.20 mmol) was dried azeotropically with 3 × 3
mL acetonitrile. During coevaporation, care should be taken to ensure
that the temperature of the flask does not rise above 30 °C to prevent
the decomposition of the tetrabutyl ammonium fluoride trihydrate.
The contents of the flask were redissolved in a small amount of
anhydrous acetonitrile, and this solution was added to a refluxing
solution of 1, 3-propanediol di-p-tosylate 6 (76.9 mg, 0.2 mmol) in
acetonitrile. The reaction progress was monitored by TLC on silica
plates [DCM: hexane (1.25:1 v/v)]. The refluxing was continued
overnight with stirring. The obtained solution was filtered, and the
filtrate was evaporated under reduced pressure. The residue was
redissolved in a small amount of mobile phase and purified using silica
gel column chromatography with a mixture of DCM and hexane
(50%) as the eluent to yield pure 7 (Scheme 1) in the form of a clear,
slightly yellowish oil.
ESI-HRMS m/z 213.0583 [M − F]+, [C10H13O3S−F]+: calcd
213.0585; high in-source fragmentation; 1H NMR (400 MHz,
CDCl3): δ = 7.80 (d, J = 8.2, 2H), 7.35 (d, J = 8.1, 2H), 4.54 (t, J
= 5.7, 1H), 4.45 (t, J = 5.6, 1H), 4.16 (t, J = 6.2, 2H), 2.45 (s, 3H),
2.14−1.94 (m, 2H).
7-(3-(4-(2-Fluoroethoxy)phenyl)propyl)-2-(furan-2-yl)-7H-
pyrazolo[4,3-e][1,2,4]triazolo [1,5-c]pyrimidin-5-amine (10a). The
synthesis of this compound was performed according to a published
procedure. The authenticity of the 10a (Scheme 2) was confirmed by
1H NMR, 13C NMR, and MS data and was in agreement with the
Methods for Chemical Synthesis and Molecular Character-
ization. Nuclear magnetic resonance spectra were recorded on a
Bruker Avance 500 spectrometer [(1H NMR (500 MHz), 13C NMR
(125 MHz)], or a Varian Oxford 400 MHz spectrometer [1H NMR
(400 MHz), 13C NMR (100 MHz)]. Chemical shifts are reported as δ
values and coupling constants are presented in hertz (Hz). Chemical
shifts for 13C NMR spectra are reported in ppm relative to the solvent
peak.
UHPLC-HRMS was used to assess the exact molecular weight of
the reference compounds ([18F]-10a and [18F]-10b) and intermediate
fluorosynthons ([18F]-5 and [18F]-7). The HPLC method was used to
calculate the purity of the compounds. Purities of all compounds were
published values.47
7-(3-(4-(3-Fluoropropoxy)phenyl)propyl)-2-(furan-2-yl)-7H-
pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (10b). The
selective fluoropropylation reaction described in the literature was
slightly modified to generate authentic nonradioactive 10b (Scheme
2).54 Our procedure differed in the fluoroalkylating agent; we used 7
instead of 1-bromo-3-fluoropropane.
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The phenol precursor 9 was dried azeotropically with 3 × 8 mL
anhydrous toluene and then suspended in anhydrous MeOH (3 mL),
and Cs2CO3 (110 mg, 0.34 mmol) and 3-fluoropropyltosylate 7 (79
mg, 0.34 mmol) were added. The reaction mixture was refluxed for 16
h with continuous stirring. The conversion was monitored by TLC
(14% acetonitrile in chloroform). The crude mixture was then
concentrated in vacuo, and the crude product was redissolved and
purified by preparative TLC with 14% acetonitrile in chloroform as a
mobile phase to furnish 7.9 mg of product. The product was
contaminated with trace silica gel. To remove the silica gel impurities,
the sample was redissolved in the mobile phase, passed through a 0.45
μm polytetrafluoroethylene (PTFE) syringe filter, and concentrated to
provide 7.4 mg (25%) of 10b (Scheme 2) as a white solid.
Independent quality control was performed on a Waters (Milford,
MA) Acquity Ultraperformace LC quaternary solvent manager
coupled to a tunable, dual-wavelength Ultraviolet/Visible (UV/vis)
detector and a radioactivity detector (Berthold Flowstar LB 513). The
radioactive product (10 μL) was injected into a Waters 3.0 × 50 mm
i.d., 1.7 μM Ethylene-Bridged Hybrid (BEH) shield RP18 column and
eluted using 40% acetonitrile at a flow rate of 0.8 mL/min. The
instrument and column temperature were set at 254 nm and 35 °C,
respectively. The retention time for [18F]10b was 3.2 min, and the
retention time for [18F]10a was 1.9 min.
Ligand Metabolism. In Silico Metabolite Analysis. The
SMARTCyp web service (version 2.4.2) was used to predict which
sites in the molecule are most vulnerable to CYP450 metabolism.56
SMARTCyp has been shown to be valid for the metabolism of the
major isoforms 1A2, 3A4, 2A6, 2B6, 2C8, 2C19, and 2E1. Additionally,
it is applicable to specific models for the 2C9 (CYP2C9) and 2D6
(CYP2D6) isoforms.56
SMARTCyp only uses the 2D structure of a compound as input,
and atoms are scored based on their propensity to undergo
metabolism, which is in turn calculated based on energy and
accessibility factors. The energy required for oxidation at each atom
is computed by fragment matching toward the SMARTS patterns. The
accessibility is approximated as the relative topological distance of an
atom from the center of the molecule, and the final score is computed
as score = energy − 8 × accessibility.56
ESI-HRMS m/z 436.1899 [M + H]+, C22H22N7O2F.H+: calcd
436.1897; 1H NMR (500 MHz, CDCl3): δ 8.24 (s, 1H), 7.67 (s, 1H),
7.29 (d, J = 5.8 Hz, 2H), 7.12 (d, J = 8.0 Hz, 2H), 6.83 (d, J = 8.1 Hz,
2H), 6.64 (s, 1H), 5.98 (s, 2H), 4.70 (t, J = 5.6 Hz, 1H), 4.61 (t, J =
5.5 Hz, 1H), 4.40 (t, J = 6.7 Hz, 1H), 4.08 (t, J = 5.9 Hz, 1H), 2.64 (t,
J = 7.4 Hz, 2H), 2.37−2.04 (m, 4H); 13C NMR (125 MHz, CDCl3): δ
157.08, 149.15, 147.83, 145.49, 144.87, 144.64, 133.26, 132.03, 129.38,
114.40, 112.74, 112.04, 97.26, 81.47, 80.16, 63.53, 46.97, 31.98, 31.19,
30.53, 30.38, 29.71.
Radiochemistry. [18F]-10a and [18F]-10b were prepared by a
two-pot radiosynthetic method, using the intermediate fluorosynthons
[18F]-5 and [18F]-7 (Scheme 3, Table 2).
No-carrier added aqueous [18F] fluoride was produced by the
irradiation of [18O] water via the 18O (p,n) 18F nuclear reaction using a
Scanditronix MC-17 biomedical cyclotron. The [18F] fluoride solution
from the target was trapped onto a preactivated Sep-Pak light Accel
plus QMA anion-exchange cartridge to recover the 18O-enriched
water. The [18F]fluoride was then eluted from the column with 1 mL
of potassium carbonate solution (1 mg/mL) into a glass conical vial
containing 15 mg of Kryptofix [2.2.2]. To this mixture, 1 mL of
acetonitrile (Rathburn, The Netherlands) was added, and the solvents
were evaporated to dryness azeotropically at 130 °C. The [18F]KF/
Kryptofix [2.2.2] complex was dried 3 times by the addition of 0.5 mL
acetonitrile, and the solvent mixture was evaporated.
A solution of 6 or 1,2-ethanediol di-p-tosylate 11 (4−5 mg) in
acetonitrile (0.5 mL) was added to the conical vial, and the vial was
heated on a heating block to 125 °C for 10 min under sealed
conditions and then cooled to room temperature to generate the crude
intermediate. The cooled reaction mixture was diluted with 4 mL
hexane/diethyl ether (3:1) and loaded onto a Sep-Pak Silica Plus
column (Waters). The cartridge was then eluted with 10 mL of
hexane/diethyl ether (3:1), and the eluate captured in a counting vial
was evaporated on a rotavap (Buchi HB-140) to obtain the desired
pure [18F]fluorosynthon [18F]-5 or [18F]-7.
Phenol precursor 9 (1.5 mg) in 0.3 mL acetonitrile and 10 μL of
40% aqueous tetrabutylammonium hydroxide solution were added to
the residual solution of 3-[18F]-fluoropropyltosylate [18F]-7 or 2-[18F]-
fluoroethyltosylate [18F]-5. The reaction vials were closed and
maintained at 115 °C for 15 min on a heating block. After cooling
to room temperature, the reaction mixture was diluted with 0.6 mL of
HPLC mobile phase and injected onto a semipreparative Phenomenex
Prodigy ODS C-18 RP-HPLC column (5 μm, 10 × 250 mm)
connected to a UV-spectrometer (Waters 486 tunable absorbance
detector) set at 254 nm and a Bicron Frisk-Tech radiation detector.
The HPLC column was then eluted with 45% acetonitrile and 55%
100 mM ammonium acetate at 5 mL/min to yield [18F]-10b (tR = 16
min); in the case of [18F]-10a, the elution solvent was 40% acetonitrile
and 60% 100 mM CH3COONH4, tR = 21 min. The collected HPLC
fractions containing the products were diluted with water (25 mL) and
loaded onto C-18 light Sep Pak cartridges (Waters). After trapping the
radioactivity, the columns were washed with an additional 4 mL of
water. The columns were dried with a flow of nitrogen and eluted with
1 mL of ethanol over a 0.22 μm Millex LG sterilization filter; the
products were collected in a 25 mL sterile vial (Mallinckrodt
pharmaceuticals, The Netherlands). The products were diluted with
saline (4 mL), and the formulated tracers were submitted for quality
control.
Human Liver Microsomal Metabolite Analysis. Human liver
microsomes (HLM) from 150 mixed-gender donors and a NADPH-
regenerating system consisting of two solutions (NADP plus glucose-
6-phosphate and glucose-6-phosphate dehydrogenase) were purchased
from BD Biosciences. Dulbecco’s Phosphate-Buffered Saline (DPBS),
pH 7.4, was procured from Life Technologies.
Nonradioactive compounds 8 and 10b were incubated with HLM.
The metabolic reactions were initiated by the addition of NADPH. At
different time points (0, 5, 15, 30, 45, 60, and 90 min), 100 μL of the
solution was taken. After quenching with cold acetonitrile and
centrifugation at 17250g, the supernatant solution (5 μL) was injected
into a UHPLC/Q-ToF-MS. Incubations without NADPH, micro-
somes, and test compound were conducted as negative controls to
characterize the nonmetabolism related degradation of the test
compound. Furthermore, verapamil was included as a positive control
because it is known to be metabolized by human liver microsomes.
MetaboLynx (Waters) was used to assist in the identification of
metabolites.
UHPLC/Q-TOF-MS Method. Data acquisition was performed using
Waters (Milford, MA) Acquity Ultraperformace LC quaternary solvent
manager. The samples were injected onto a Waters 3.0 × 50 mm
internal diameter (i.d.), 1.7 μM BEH shield RP 18 column and eluted
using a 6 min gradient starting from 0.1% formic acid in 98% water for
5 min, then with a 100% acetonitrile solution for 0.5 min, and finally
with 0.1% formic acid in 98% water at a flow rate of 0.6 mL/min and a
column temperature of 35 °C. The mass spectrometer was operated in
electrospray positive ionization mode with an extended dynamic range
and resolution mode analyzer; the machine settings were 0.5 kV
capillary voltage, 45 V sampling cone, 4 V extraction cone, and 150
and 500 (°C) source and desolvation temperature, respectively.
Leucine enkephalin was used as the lock mass (m/z 556.2771) at a
concentration of 500 pg/μL.
LogD7.4 Measurement. After tracer elution from a C-18 light Sep
Pak column, 500 mL of eluate (octanol) was mixed with an equal
volume of 1 M phosphate buffer (pH 7.4) and vigorously vortexed for
1 min and centrifuged (10 min, 17250g). Three 100 μL aliquots were
drawn from the corresponding n-octanol and aqueous phases. The
radioactivity in each phase was counted (Compugamma 1282 CS,
LKB-Wallac, Finland). The experiments were performed in triplicate
for each tracer batch; the average logD7.4 value is reported.
In Vitro Ligand Stability Test. The in vitro stability tests were
performed by dissolving the formulated tracers in PBS, saline, rat
plasma, and human plasma and incubating these solutions at 37 °C.
After 1 and 2 h of incubation, the solutions were analyzed by radio-
TLC (Rf [18F]-10a = 0.4 and [18F]-10b = 0.5, 14% acetonitrile in
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chloroform). The rat and human plasma samples were deproteinized
by adding 3 volumes of acetonitrile and centrifuged (5 min at 17250g)
before they were used for analysis. After elution with the mobile phase,
the TLCs were dried and placed on phosphor storage plates, which
were later scanned with a Cyclone imaging system (PerkinElmer). The
percentage of conversion as a function of incubation time was
calculated by ROI analysis using Optiquant (version 3.00).
In Vivo and In Vitro Selectivity. Nonradioactive A2AR-selective
12 [1 mg/kg, 50% dimethylacetamide (DMA): saline (v/v)] or vehicle
(1 mL/kg, 50% DMA:saline)32 were intraperitoneally administered 5−
6 min prior to the intravenous injection of [18F]tracers. In the in vitro
ARG experiments, 2 μM (0.77 mg) 12 was used as a blocking agent.
In Vitro ARG Experiment. Isolated frozen brains of young (10−
12 weeks of age; 300−350 g body weight) male Sprague−Dawley rats
(Harlan, The Netherlands) were cut into two halves along the sagittal
symmetry plane. Each half was mounted on paper slides with its lateral
side up using Tissue-Tek fixing gel (Sakura, The Netherlands). After
fixing, the brain was cut sagittally into 20 μm thick cryostat sections
using a Leica CM 1950 cryostat (Leica Biosystems, The Netherlands)
at −15 °C. The slices were thaw-mounted onto starfrost (76 × 26 mm,
Waldemar Knittel, Germany) adhesive precoated slides, air-dried for
30−40 min and stored at −80 °C until they were used (within 1
week).
The slices were brought to room temperature and preincubated for
15 min in an assay buffer (50 mM Tris-HCl at pH 7.5 with 10 mM
MgCl2 and 1% bovine serum albumin). After preincubation, the slides
were placed into jars with an assay buffer containing an approximately
5 nM radioligand [[18F]-10a: 4.5 1.5 nM (n = 3), 1.2 MBq and
[18F]-10b: 5.1 1.8 nM (n = 5), 1.02 MBq]. To test the specificity of
the binding, a 2 μM compound, 12, was added to the buffer in one of
the jars. The slices were incubated for 90 min at 37 °C. After
incubation, the slices were washed for 5 min with ice-cold 0.01%
Triton X in PBS. They were dipped in ice-cold water for 30 s to
remove buffer salts and dried under a stream of air at room
temperature. After drying, the slices were placed on phosphor storage
screens for 8−10 h. The screens were then read using a Cyclone
imaging system (Packard Instrument Co.). Optiquant (version 3.00)
was used to quantify radioactivity. Regions of interest (ROIs) were
drawn manually on the striatum and cerebellum. The regional uptake
of radioactivity was measured and expressed as digital light units
(DLU)/mm2.
In Vivo Studies. Animals and Study Design. The animal
experiments were carried out in compliance with the Law on Animal
Experiments of The Netherlands. The institutional animal ethics
committee approved the protocols. Male outbred Wistar-Unilever rats
were obtained from Harlan (The Netherlands). The animals were
housed in Macrolon cages (38 × 26 × 24 cm), maintained in a 12 h
light−dark regime, and fed with standard laboratory chow (RMH-B,
The Netherlands) and tap water ad libitum. After arrival, the rats were
allowed to acclimatize for at least 7 days. For each tracer, the animals
were divided into two groups, as follows. Group 1, vehicle-controls
([18F]-10b (n = 6): body weight = 295 19 g and injected dose =
0.21 0.11 nM; [18F]-10a (n = 5): body weight = 312 14 g and
reconstructed in 25 time frames (8 × 30, 3 × 60, 2 × 120, 2 × 180, 3 ×
300, 5 × 600, 1 × 480, and 1 × 960 s) using a 2D ordered subsets
expectation maximization (OSEM) algorithm (4 iterations, zoom
factor, 2). The reconstructed images were smoothed with a 3D
Gaussian filter [1.35 mm full width at half-maximum (fwhm)].
During the scan, arterial blood samples (volume 0.1−0.15 mL) were
drawn using a standard protocol (at 0, 5, 10, 15, 20, 30, 45, 60, 75, and
90 s and 2, 3, 5, 7, 10, 15, 30, 60, and 90 min after injection). After
collecting 25 μL of whole blood, plasma (25 μL) was acquired from
the remainder of the blood samples by a short centrifugation (5 min at
1000g). The radioactivity in 25 μL of plasma and whole blood was
counted on a Compugamma γ-counter (1282 CS, LKB-Wallac, Turku,
Finland), and the count statistics were then used as an arterial input
function. The heart rates and blood oxygenation of the experimental
animals were continuously monitored using pulse oximeters
throughout the scanning procedure (Nonin Zevenaar, The Nether-
lands).
MicroPET Data Analysis. Time activity curves (TACs) for the
frontal cortex, striatum, midbrain, cerebellum, and hippocampus were
determined using Inveon Research Workplace (Siemens Medical
Solutions, Knoxville, TN). The summed PET data from each animal
were coregistered to an MRI template of the rat brain with predefined
volumes of interest (VOIs). Translation, rotation, and scaling were
adjusted to visually optimize the fusion of the images. The VOIs were
transferred from the MRI template to the PET data, and regional
TACs were generated. Standardized uptake values (SUVs) were
plotted as a function of time, using body weights and injected doses.
Ex Vivo Biodistribution. After the PET-scan, the animals were
sacrificed by the extirpation of the heart. Blood was collected from the
animals, and plasma and a cell fraction were obtained from the sample
by a short centrifugation (5 min at 1000g). Several tissues were excised
and weighed. The radioactivities in the tissue samples and in a sample
of tracer solution (infusate) were measured using a calibrated gamma
counter. The data were expressed as the SUV.
In Vivo Metabolite Analysis. Plasma samples taken at intervals of
2, 5, 10, 15, 30, 60, and 90 min were used for metabolite analysis.
Protein was removed by adding 3 volumes of acetonitrile followed by
centrifugation (5 min at 17250g). Samples (2.5 μL) of the supernatant
and infusate (internal standard, diluted 50 to 100 times) were loaded
onto a TLC plate. After development with 15% acetonitrile in
chloroform, the plate was dried and placed on a phosphor screen
which was later read by the Cyclone system. ROIs were drawn
manually on the parent and metabolite spots. The concentration of the
parent tracer was expressed as the percentage of total radioactivity in
the acetonitrile extract. Optiquant was used for radioactivity
quantification.
Statistical Analysis. All results are expressed as the mean SEM.
The differences between groups were examined using an unpaired
two-tailed t test. P < 0.05 was considered to be statistically significant.
AUTHOR INFORMATION
■
injected dose = 1.13
0.41 nM) and group 2, pretreated group
Corresponding Author
([18F]-10b (n = 6): body weight = 293 31 g and injected dose =
0.29 0.27 nM; [18F]-10a (n = 5): body weight = 321 15 g and
injected dose = 0.74 0.62 nM).
*Address: Department of Nuclear Medicine and Molecular
Imaging, University Medical Center Groningen, University of
Groningen, Hanzeplein 1, 9713GZ, Groningen, The Nether-
lands. Tel: +3150 3613247. Fax: +31-50-3611687. E-mail: p.h.
elsinga@umcg.nl.
Micro PET Scanning. Two animals were scanned simultaneously in
each scan session (Supine position) using a Focus 220 MicroPET
camera (CTI, Siemens, Munich, Germany). All animals were
anesthetized with isoflurane/air (induction: 5% isoflurane, later
reduced to ≤2%) and kept on electronic heating pads during the
entire study period. Cannulas were placed in a femoral artery and vein
for blood sampling and tracer injection (Harvard-style pump; 1 mL/
min). The brain was in the field of view. A transmission scan of 515 s
was made before the emission scan, using a rotating 57Co point source.
The emission data were acquired in list mode for 106 min, starting at
the moment of tracer entering the body of the first rat; the second
animal was injected 16 min later. PET data were corrected for
attenuation, scatter, random coincidences, and radioactive decay and
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was supported by De Cock Stichting and ZonMW
(Grant 91111007, UHPLC-MS-TOF). We thank Jurgen
Sijbesma, MA Khayum and Marianne Schepers for their
technical assistance.
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Journal of Medicinal Chemistry
Article
(14) Paul, S.; Elsinga, P. H.; Ishiwata, K.; Dierckx, R. A.; van Waarde,
A. Adenosine A(1) receptors in the central nervous system: Their
functions in health and disease, and possible elucidation by PET
imaging. Curr. Med. Chem. 2011, 18, 4820−4835.
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ABBREVIATIONS USED
■
A2ARs, adenosine A2A receptors; ARG, autoradiography; BBr3,
boron tribromide; BBB, blood-brain-barrier; BEH, ethylene-
bridged hybrid; cAMP, cyclic adenosine monophosphate;
CDCl3, deuterated chloroform; CSPTC, cortico-striato-pal-
lido-thalamocortical; D2Rs, dopamine d2 receptors; ECL,
extracellular loop; g, Earth’s gravitational force; GOLD, a
molecular docking software; Gs, heterotrimeric G protein;
HLM, human liver microsome; Koff, dissociation rate constant
LogD7.4, octanol/buffer (pH 7.4) distribution coefficient;
MicroPET, small-animal PET; nM, nanomolar; PTFE,
polytetrafluoroethylene; QMA, quaternary ammonium anion;
Q-Tof-Ms, quadrupole-time-of-flight-mass spectrometer; radio-
TLC, radio-thin layer chromatography; ROIs, regions of
interest; RP18, reverse phase 18; RP-HPLC, reverse-phase-
high performance liquid chromatography; SUV, standardized
uptake value; SEM, standard error of mean; TM’s, trans-
membranes; TACs, time-activity curves; VOIs, volumes of
interest
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