Discovery of a fluorinated 4-oxo-quinoline derivative as a potential positron emission tomography radiotracer for imaging cannabinoid receptor type 2

Article abstract of DOI:10.1111/jnc.13716

The cannabinoid receptor type 2 (CB2) is part of the endocannabinoid system and has gained growing attention in recent years because of its important role in neuroinflammatory/neurodegenerative diseases. Recently, we reported on a carbon-11 labeled 4-oxo-

Full text of DOI:10.1111/jnc.13716

JOURNAL OF NEUROCHEMISTRY
|
2016
doi: 10.1111/jnc.13716
,
,
*Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland
†Neuromuscular Diseases Unit/ALS Clinic, Kantonsspital St. Gallen, St. Gallen, Switzerland
‡Department of Nuclear Medicine, University Hospital Zurich, Zurich, Switzerland
Abstract
The cannabinoid receptor type
whereas 55% intact parent compound was found in vivo in rat
2
(CB2) is part of the
blood plasma at 10 min post injection. In vitro autoradio-
graphic studies with CB2-positive rat spleen tissue revealed
high and blockable binding which was confirmed in in vivo
displacement experiments with rats by dynamic PET imaging.
Ex vivo biodistribution studies confirmed accumulation of [18F]
RS-126 in rat spleen with a specificity of 79% under blocking
conditions. The moderate elevated CB2 levels in LPS-treated
mice brain did not permit the detection of CB2 by [18F]RS-126
using PET imaging. In summary, [18F]RS-126 demonstrated
high specificity toward CB2 receptor in vitro and in vivo and is
a promising radioligand for imaging CB2 receptor expression.
endocannabinoid system and has gained growing attention
in recent years because of its important role in neuroinflam-
matory/neurodegenerative diseases. Recently, we reported on
a carbon-11 labeled 4-oxo-quinoline derivative, designated
RS-016, as a promising radiotracer for imaging CB2 using
PET. In this study, three novel fluorinated analogs of RS-016
were designed, synthesized, and pharmacologically evalu-
ated. The results of our efforts led to the identification of N-(1-
adamantyl)-1-(2-(2-fluoroethoxy)ethyl)-8-methoxy-4-oxo-1,4-
dihydroquinoline-3-carboxamide (RS-126) as the most potent
candidate for evaluation as a CB2 PET ligand. [18F]RS-126
was obtained in ≥ 99% radiochemical purity with an average
specific radioactivity of 98 GBq/lmol at the end of the
radiosynthesis. [18F]RS-126 showed a logD7.4 value of 1.99
and is stable in vitro in rat and human plasma over 120 min,
Keywords: biodistribution,
CB2
ligands,
fluorine-18
radiolabeling, in vitro autoradiography, neuroinflammation,
positron emission tomography.
J. Neurochem. (2016) 10.1111/jnc.13716
Although the effects of cannabinoids have been known for
centuries, the functional receptors (CB1 and CB2) were
discovered only in the past decades through investigations
on potential targets for cannabidiol and D9-tetrahydrocan-
nabinol, the two pharmacologically most active ingredients
of the hemp plant Cannabis sativa (Matsuda et al. 1990;
Munro et al. 1993). Both CB1 and CB2 belong to the class
A (rhodopsin family) G protein-coupled receptor family.
The CB1 receptors are abundantly expressed in the central
nervous system (CNS) and their function has been thor-
oughly investigated. The CB2 receptors are found predom-
inantly in cells of the immune system, e.g. in the spleen,
thymus or peripheral blood mononuclear cells and have very
low to undetectable expression levels in the CNS under
basal conditions (Galiegue et al. 1995). Cannabinoid recep-
tors have gained attention as potential therapeutic targets for
Received April 13, 2016; revised manuscript received June 8, 2016;
accepted June 9, 2016.
Address correspondence and reprint requests to Dr Linjing Mu or Prof.
Dr Simon M. Ametamey, Institute of Pharmaceutical Sciences, ETH
Zurich, 8093 Zurich, Switzerland. E-mails: linjing.mu@usz.ch, simon.
ametamey@pharma.ethz.ch
Abbreviations used: CB2, cannabinoid receptor type 2; DAST,
diethylaminosulfur trifluoride; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate; HRMS, high-resolution mass
spectrometry; PET, positron emission tomography; TSPO, translocator
protein.
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716
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R. Slavik et al.
the treatment of neurodegenerative diseases, cancer, obesity,
inflammatory bowel disease, and pain (Di Marzo 2009).
CB1 agonists and antagonists are medically used to enhance
or reduce appetite, relief pain, avoid vomiting and muscle
spasticity, a major limitation for the therapeutic develop-
ment of compounds that directly activate CB1 receptors is
unwanted psychotropic effects (Pertwee 2009; Volkow
et al. 2014). Because of these undesirable effects, growing
attention is being paid to the second cannabinoid receptor
CB2. The high expression of CB2 receptor in cells and
tissue of the immune system as well as the enhancement of
its expression following inflammatory insults (Wright et al.
2005; Mukhopadhyay et al. 2006), suggests that CB2
receptor selective ligands might be effective in modulating
inflammation. Initial studies revealed that activation of CB2
receptor reduced the production of pro-inflammatory
molecules in a number of murine and human cell types
in vitro (Merighi et al. 2012). Selective CB2 ligands have
the potential to be used to treat pain, inflammation,
osteoporosis, CB2-expressing malignant gliomas, tumors
of immune origin, and various immunological disorders.
CB2 activation may also produce analgesic effects without
the CNS-mediated side effects encountered with CB1
receptor agonists/antagonists.
Positron emission tomography (PET) is a non-invasive
biomedical imaging technique that enables quantification
and visualization of biochemical and physiological pro-
cesses by monitoring the time-dependent distribution of
trace amount of molecules labeled with positron-emitting
isotopes. PET imaging can provide information about
receptor localization and density and has proven to be a
powerful technique in neurological research (Halldin et al.
2001; Ametamey et al. 2008; Honer et al. 2014; Piel et al.
2014). The development of radioligands for non-invasive
PET imaging of molecular targets in the central and
peripheral nervous systems is a demanding task. This is
particularly true for the imaging of neuroinflammation and
neurodegeneration. Historically, nearly all neuroinflamma-
tion PET radiotracer research was related with the 18-kDa
translocator protein (TSPO, formerly called peripheral
benzodiazepine receptor). Despite the great efforts that
have been made in the development of TSPO radioligands
(Boutin et al. 2007; Endres et al. 2009; Van Camp et al.
2010; Damont et al. 2015; Vicidomini et al. 2015), their
use in PET imaging of neuroinflammation remains chal-
lenging. TSPO radioligands exhibit a high non-specific
binding, have complex kinetics and variable concentrations
in plasma-free fractions across human clinical cohorts
(Chauveau et al. 2008; Owen and Matthews 2011;
Turkheimer et al. 2015). The heterogeneous TSPO distri-
bution in brain tissue and polymorphisms of the TSPO gene
even hampers radiotracer affinity. Development of a PET
radiotracer that binds to a different target that is enhanced
in neuroinflammation is an attractive alternative. One such
alternative to TSPO is CB2. CB2 expression in microglia is
up-regulated during activation. Neuroprotective effects of
CB2 agonists are associated with suppression of microglia
activation via inhibiting the release of neurotoxic factors
and by decreasing neuronal cell damage in cell or tissue
culture models (Eljaschewitsch et al. 2006). Increased
expression of CB2 under neuroinflammatory conditions
was found in human brain (Benito et al. 2008). Prominent
CB2 up-regulation was reported in brain tissues affected by
multiple sclerosis, amyotrophic lateral sclerosis, Down
syndrome, and Alzheimer’s disease (Benito et al. 2007;
Shoemaker et al. 2007; Solas et al. 2013). CB2-specific
radiotracer can be used as a biomarker of neuroinflamma-
tion in the early preclinical stages of Alzheimer’s disease,
when no significant neuronal loss has yet developed
(Savonenko et al. 2015). A number of PET radioligands
have been evaluated, but most of the ligands have been
plagued with either high non-specific binding, low affinity
and selectivity, rapid metabolism, or inappropriate pharma-
cokinetics (Evens et al. 2008, 2009, 2012; Horti et al.
2010; Vandeputte et al. 2011; Ruhl et al. 2012; Turkman
et al. 2012; Gao et al. 2014). Recently, we reported a novel
carbon-11 radiolabeled tracer [11C]RS-016 for imaging CB2
(Slavik et al. 2015b). High binding to CB2 was observed in
CB2-positive tissues (spleen) in rodents and postmortem
amyotrophic lateral sclerosis patient spinal cord tissues in
in vitro autoradiography experiments. Biodistribution data
confirmed the high and specific uptake of [11C]RS-016 in
rat spleen. Drawback of this tracer is the short physical
half-life (20 min) of carbon-11 that limits its application to
only centers with an on-site cyclotron/radiochemistry facil-
ity. Compared to 11C, the longer physical half-life of 18F
(110 min) allows for multiple PET scanning of several
individuals from a single preparation, and permits also the
distribution of the radiopharmaceutical to other PET
imaging centers that lack a radiochemistry facility. The
high percentage (96.7%) and relatively low energy
(635 keV) of positron b+ emission result in a low radiation
dose to patients and high-resolution images.
Herein, we present the synthesis of three fluorinated
analogs of RS-016 namely RS-030, RS-122, and RS-126
(Fig. 1), and their binding affinities toward CB2. The most
potent candidate, RS-126, was radiolabeled with fluorine-18
and its potential as a CB2 PET ligand was evaluated both
in vitro and in vivo.
Materials and methods
General
All reagents and solvents were purchased from Sigma-Aldrich
Chemie GmbH (Steinheim am Albuch, Germany), Merck (Darmstadt,
Germany), Acros Organics (Belgium), ABCR GmbH (Karlsruhe,
Germany) or Fluka (Buchs, Switzerland). All chemicals were used as
supplied without further purification. 1H and 13C NMR spectra were
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716

PET radiotracer for imaging CB2 receptor
3
Fig. 1 Three novel fluorinated RS-016
analogs.
obtained on a Bruker Avance FT-NMR spectrometer (400 MHz)
(Bruker BioSpin AG, Fällanden, Switzerland). Chemical shifts are
given in delta (d) units, in ppm relative to tetramethylsilane (0 ppm).
1 h. After cooling to ambient temperature, the mixture was filtered and
the precipitates were washed with diphenylether (10 mL) and hexane
(3 9 10 mL). After recrystallization from ethanol (150 mL), the
precipitates were collected by filtration and dried under reduced
pressure to give 2 (7.68 g, 70%) as a gray powder. 1H NMR
(400 MHz, dimethylsulfoxide, DMSO): d 11.92 (d, J = 4.2 Hz, 1H),
8.34 (d, J = 6.0 Hz, 1H), 7.70 (dd, J = 7.5 Hz, 1.9 Hz, J2 = 7.5 Hz,
1H), 7.36–7.30 (m, 2H), 4.21 (q, J = 7.1 Hz, 2H), 4.00 (s, 3H), 1.27
(t, J = 7.1, 3H). 13C NMR (100 MHz, DMSO): d 173.2, 164.2, 148.7,
144.0, 129.3, 128.1, 124.6, 116.7, 112.2, 110.0, 59.6, 56.3, 14.3.
HRMS calcd for C13H13NNaO4 (M+Na) 270.0737, found 270.0743.
Synthesis of ethyl 1-(2-ethoxyethyl)-8-methoxy-4-oxo-1,4-dihy-
1
Multiplicities in the H-NMR spectra are described as: s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet. Coupling con-
stants (J) are reported in Hz. High-resolution mass spectrometry
(HRMS) was performed on a Bruker’s maXis (ESI-Qq-TOF-MS)
spectrometer (Bruker Daltonik GmbH, Bremen, Germany) and are
given in m/z. ClogP values were calculated using the CambridgeSoft
Software ChemDraw 13.0 (PerkinElmer, Cambridge, MA, USA). For
purification of the radiolabeled product, a semi-preparative Knauer
Smartline 1050 HPLC system was used with an ACE C18-300
column (250 9 10 mm, 5 lm) with an isocratic solvent system 0.1%
H3PO4 in H2O (60%) and CH3CN (40%) at a flow rate of 4 mL/min.
For product analysis, an analytical Agilent 1100 series HPLC system
(Agilent Technologies AG, Morges, Switzerland), equipped with UV
multi-wavelength detector and a GabiStar radiodetector (Raytest) was
used with an ACE C18-AR column (50 9 4.6 mm, 3 lm) with the
following conditions: 0.1% trifluoroacetic acid in H2O (solvent A),
CH3CN (solvent B); 0.0–3.0 min, 30% B; 3.1–13.0 min, 30–95% B;
13.1–15 min, 95% B at a flow rate of 1 mL/min.
droquinoline-3-carboxylate (3). To
a solution of 2 (1.2 g,
4.85 mmol) in N, N-dimethylformamide (15 mL), potassium
carbonate (1.34 g, 9.71 mmol) and 1-bromo-2-ethoxyethane
(1.095 mL, 9.71 mmol) were added. The reaction mixture was
heated at 90 °C for 4 h, cooled to RT (23–25°C) and then aq. HCl
(0.2 M, 50 mL) was added. The mixture was extracted with CH2Cl2
(3 9 5 mL) and the combined organic layers dried over MgSO4.
Solvents were removed under reduced pressure and the residue was
purified over silica gel using hexane/EtOAc (1 : 1) to give 3
(1.16 g, 75%) as a yellowish oil. 1H NMR (400 MHz, CDCl3): d
9.19 (s, 1H), 7.96 (dd, J = 8.3 Hz, 1.0 Hz, 1H), 7.49 (t,
J = 8.3 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1.0 Hz, 1H), 4.45 (q,
J = 7.1 Hz, 2H), 4.40–4.38 (m, 2H), 4.08 (s, 3H), 3.85–3.83 (m,
2H), 3.57 (q, J = 7.0 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H), 1.20 (t,
J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3): d 165.1, 164.1,
155.4, 150.8, 142.8, 126.9, 125.1, 115.2, 115.1, 109.8, 75.3, 69.6,
66.7, 61.5, 56.1, 15.1, 14.3. HRMS calcd for C17H22NO5 320.1492
(M+H), found 320.1488.
Synthesis of 1-(2-ethoxyethyl)-8-methoxy-4-oxo-1,4-dihydroqui-
noline-3-carboxylic acid (4). Aq. NaOH (10%, 100 mL) was added
to 3 (1.5 g, 4.7 mmol). The reaction mixture was refluxed for 3 h.
After cooling to ambient temperature, pH was adjusted to two using
conc. HCl and the precipitates were filtered and washed with water
(10 mL) and diethylether (10 mL). Recrystallization from EtOH
(80 mL) gave 4 (1.16 g, 85%) as a yellowish solid. 1H NMR
(400 MHz, CDCl3): d 14.95 (s, 1H), 8.18 (dd, J = 8.1 Hz, 1.4 Hz,
1H), 7.49 (t, J = 8.1 Hz, 1H), 7.29 (dd, J = 8.1 Hz, 1.4 Hz, 1H),
4.82 (t, J = 4.9 Hz, 2H), 4.00 (s, 3H), 3.76 (t, J = 4.9 Hz, 2H), 3.40
Chemistry
Synthesis of diethyl 2-(((2-methoxyphenyl)amino)methylene)mal-
onate (1). A mixture of 2-anisidine (6.36 g, 51.6 mmol) and diethyl
2-(ethoxymethylene)malonate (11.17 g, 51.6 mmol) was stirred and
heated in an oil bath at 110 °C for 1 h. After cooling to ambient
temperature, the crude mixture was recrystallized from hexane
(50 mL) to give 1 (13.36 g, 88%) as a yellowish solid. 1H NMR
(400 MHz, CDCl3): d 11.12 (d, J = 14.0 Hz, 1H), 8.57 (d,
J = 14.0 Hz, 1H), 7.25 (m, 1H), 7.12–7.08 (m, 1H), 7.00–6.97
(m, 1H), 6.95–6.92 (m, 1H), 4.33 (q, J = 7.1 Hz, 2H), 4.25 (q,
J = 7.1 Hz, 2H), 3.94 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H), 1.33 (t,
J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3): d 168.5, 166.0,
150.4, 148.9, 128.8, 124.8, 121.2, 120.9, 119.1, 114.3, 111.2, 93.8,
60.3, 60.0, 55.9, 14.4, 14.3. HRMS calcd for C15H19NNaO5
(M+Na) 316.1155, found 316.1164.
Synthesis of ethyl 8-methoxy-4-oxo-1,4-dihydroquinoline-3-car-
boxylate (2). Compound 1 (13.00 g, 44.3 mmol) was dissolved in
diphenylether (70 mL). The reaction mixture was heated at 250 °C for
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716

4
R. Slavik et al.
(q, J = 7.0 Hz, 2H), 1.09 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
CDCl3): d 152.0, 126.5, 119.3, 115.5, 69.0, 66.8, 59.9, 56.5, 15.0.
HRMS calcd for C15H18NO5 (M+H) 292.1179, found 292.1175.
Synthesis of 1-(2-ethoxyethyl)-N-(3-hydroxyadamantan-1-yl)-8-
methoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide (RS-028). To
a solution of 4 (63 mg, 0.216 mmol) in DMF (1.5 mL) was added
di-isopropylethylamine (0.09 mL, 0.515 mmol). 2-(1H-benzotria-
zol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU,
164 mg, 0.433 mmol) was added portion wise and the reaction
mixture is stirred at 23–25°C for 30 min, then 3-hydroxy-1-
aminoadamantane (36.2 mg, 0.216 mmol) was added. The reaction
mixture was stirred at 23–25°C for 3 h. The mixture was diluted
with ethyl acetate (EtOAc, 50 mL) and washed with water
(3 9 15 mL), followed by diluted HCl (0.5M, 15 mL), water
(15 mL) and then brine (20 mL). The organic solvent was
evaporated under reduced pressure and the residue was purified
with flash chromatography using CH2Cl2/MeOH (50 : 1 to 20 : 1)
to give RS-028 (80 mg, 84%) as a white solid. 1H NMR (400 MHz,
CDCl3): d 10.04 (s, 1H), 8.64 (s, 1H), 8.16 (dd, J = 8.1 Hz, 1.4 Hz,
1H), 7.39 (t, J = 8.0 Hz, 1H), 7.19 (dd, J = 8.1 Hz, 1.2 Hz, 1H),
4.74 (t, J = 5.4 Hz, 2H), 3.97 (s, 3H), 3.75 (t, J = 5.4 Hz, 2H), 3.42
(q, J = 7.0 Hz, 2H), 2.31–2.04 (m, 9H), 1.78–1.62 (m, 5H), 1.42 (s,
1H), 1.11 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3): d
150.9, 114.3, 69.5, 59.0, 56.3, 49.3, 44.2, 40.5, 35.1, 30.7. HRMS
calcd for C25H33N2O5 441.2384 (M+H), found 441.2382.
1.78–1.67 (m, 12H), 1.18 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
CDCl3): d 149.11, 125.62, 123.36, 119.80, 69.37, 67.81, 55.34,
41.76, 36.53, 31.93, 29.70, 29.53, 29.36, 14.62. HRMS calcd for
C24H31N2O4 (M+H) 411.2278, found 411.2281.
Synthesis of N-(1-adamantanyl)-1-(2-ethoxyethyl)-8-(2-fluor-
oethoxy)-4-oxo-1,4-dihydroquinoline-3-carboxamide (RS-122). To
a solution of 5 (20 mg, 0.05 mmol) and cesium carbonate (24 mg,
0.075 mmol) in DMF (1 mL) was added 2-fluoroethyl 4-methyl-
benzenesulfonate (13 lL, 0.075 mmol). The reaction mixture was
stirred at 23–25°C for 24 h. The mixture was diluted with aq. HCl
(0.2 M, 30 mL) and extracted with CH2Cl2 (3 9 5 mL). The
combined organic layers were washed with brine (20 mL) and dried
over MgSO4. Solvents were removed under reduced pressure and
the residue was purified over silica gel using CH2Cl2/MeOH
1
(100 : 1) to give RS-122 (18 mg, 79%) as a white solid. H NMR
(400 MHz, CDCl3): d 9.88 (s, 1H), 8.68 (s, 1H), 8.21 (dd,
J = 8.0 Hz, 1.4 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.15 (dd,
J = 8.0 Hz, 1.2 Hz, 1H), 4.90–4.88 (m, 1H), 4.78–4.76 (m, 3H),
4.40–4.38 (m, 1H), 4.33–4.31 (m, 1H), 3.78 (t, J = 5.3 Hz, 2H),
3.41 (q, J = 7.0 Hz, 2H), 2.19–2.11 (m, 9H), 1.77–1.68 (m, 6H),
1.10 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3): d 151.2,
124.9, 120.1, 114.9, 82.0, 80.3, 69.5, 68.7, 68.5, 66.7, 58.9, 51.6,
41.8, 36.6, 29.6, 15.0. HRMS calcd for C26H34FN2O4 (M+H)
457.2497, found 457.2495.
Synthesis of 8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (6). To 2 (2.00 g, 8.09 mmol) was added aq. NaOH (10%,
140 mL). The reaction mixture was heated to reflux and stirred for
3 h. The pH was adjusted to 2 using conc. HCl and the precipitates
were filtered and washed with water (15 mL) and petroleum ether 60/
90 (20 mL). The residue was dissolved in EtOH (150 mL) and
heated to reflux and stirred for 30 min. The mixture was cooled down
slowly; the precipitates were filtered and dried in vacuum to give 6
(1.67 g, 94%) as a slight gray powder. 1H NMR (400 MHz, DMSO):
d 15.32 (s, 1H), 12.99 (s, 1H), 8.57 (s, 1H), 7.83 (dd, J = 8.0 Hz,
1.4 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.49 (dd, J = 8.0 Hz, 1.2 Hz,
1H), 4.06 (s, 3H). 13C NMR (100 MHz, CDCl3): d 178.2, 166.2,
149.0, 143.9, 126.4, 125.3, 116.0, 113.5, 107.9, 56.6. HRMS calcd
for C11H10NO4 (M+H) 220.0604, found 220.0600.
Synthesis of N-(1-adamantyl)-8-methoxy-4-oxo-1,4-dihydroqui-
noline-3-carboxamide (7). To a solution of 6 (220 mg, 1 mmol) in
DMF (8 mL), di-isopropylethylamine (524 lL, 3.00 mmol) was
added and the reaction mixture was stirred at 23–25°C for 30 min.
HBTU (758 mg, 2.00 mmol) was added portion wise, followed by
the addition of 1-aminoadamantane (180 mg, 1.20 mmol). The
mixture was stirred at 23–25°C for 3 h, diluted with EtOAc (50 mL)
and washed with water (3 9 15 mL), aq. HCl (0.5M, 15 mL), water
(15 mL) and then brine (20 mL). EtOAc was evaporated under
reduced pressure and the residue was purified with flash chro-
matography using hexane/EtOAc (2 : 1 to 1 : 4) to give 7 (330 mg,
94%) as a yellowish powder. 1H NMR (400 MHz, DMSO): d 12.28
(d, J = 4.3 Hz, 1H), 10.02 (s, 1H), 8.58 (d, J = 4.3 Hz, 1H), 7.82
(dd, J = 7.9 Hz, 1.4 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.39 (dd,
J = 7.9 Hz, 1.4 Hz, 1H), 4.06 (s, 3H), 2.10 (s, 9H), 1.71 (s, 6H).
13C NMR (100 MHz, CDCl3): d 142.5, 124.7, 116.5, 112.0, 56.3,
50.4, 41.4, 36.0, 28.8. HRMS calcd for C21H25N2O3 353.1860
(M+H), found 353.1854.
Synthesis of 1-(2-ethoxyethyl)-N-(3-fluoroadamantyl)-8-meth-
oxy-4-oxo-1,4-dihydroquinoline-3-carboxamide (RS-030). To
ꢀ78 °C cold solution of 1-(2-ethoxyethyl)-N-(3-hydroxyadaman-
tyl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide (RS-
a
028) (11 mg, 0.023 mmol) in CH2Cl2 (0.25 mL), diethylaminosulfur
trifluoride (DAST, 6 lL, 0.045 mmol) was added. The mixture was
allowed to warm to 23–25°C and stirred for 1 h. Ice water (5 mL) was
added and the reaction was extracted with CH2Cl2 (3 9 2 mL),
washed with brine, dried over MgSO4 and solvents were removed
under reduced pressure. Crude was purified over silica gel using
CH2Cl2/MeOH (50 : 1) to give RS-030 (10 mg, 91%) as a white
solid. 1H NMR (400 MHz, CDCl3): d 10.09 (s, 1H), 8.64 (s, 1H), 8.16
(dd, J = 8.1 Hz, 1.4 Hz, 1H), 7.39 (t, J = 8.1, 1H), 7.19 (dd,
J = 8.1 Hz, 1.2 Hz, 1H), 4.74 (t, J = 5.3 Hz, 2H), 3.97 (s, 3H), 3.75
(t, J = 5.3 Hz, 2H), 3.41 (q, J = 7.0 Hz, 2H), 2.37 (d, J = 5.3 Hz,
4H), 2.17–1.85 (m, 8H), 1.65–1.55 (m, 2H), 1.12 (t, J = 7.0 Hz, 3H).
13C NMR (100 MHz, CDCl3): d 151.0, 125.3, 119.4, 114.3, 69.5,
66.8, 59.0, 56.4, 46.5, 41.9, 41.8, 40.4, 34.9, 31.1, 31.0, 15.0. HRMS
calcd for C25H32FN2O4 (M+H) 443.2341, found 443.2340.
Synthesis of N-(1-adamantyl)-1-(2-ethoxyethyl)-8-hydroxy-4-
oxo-1,4-dihydroquinoline-3-carboxamide (5). To a solution of RS-
016 (202 mg, 0.476 mmol) in DMF (5 mL) was added lithium
chloride (303 mg, 7.14 mmol). The reaction mixture was heated to
reflux and stirred overnight. After cooling to ambient temperature,
EtOAc (60 mL) was added and the mixture was washed with HCl
(0.2 M, 3 9 10 mL) and brine (15 mL). The organic layer was
dried over MgSO4 and solvents were removed under reduced
pressure. HPLC purification over a C18 column using 0.1% TFA in
water and acetonitrile (30 : 70, v : v) gave the desired product 5
(37 mg, 20%) as a yellowish powder. 1H NMR (400 MHz, CDCl3):
d 10.00 (s, 1H), 8.65 (s, 1H), 8.33 (br s, 1H), 8.14 (dd, J = 7.1 Hz,
2.5 Hz, 1H), 7.35–7.29 (m, 2H), 4.75 (t, J = 4.9 Hz, 2H), 4.00 (t,
J = 4.9 Hz, 2H), 3.61 (q, J = 7.0 Hz, 2H), 2.14–2.09 (m, 3H),
Synthesis of N-(1-adamantyl)-1-(2-(2-bromoethoxy)ethyl)-8-
methoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide (8). To
a
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716

PET radiotracer for imaging CB2 receptor
5
stirred solution of 7 (100 mg, 0.284 mmol) in DMF (2 mL), cesium
carbonate (137 mg, 0.422 mmol) and 1-bromo-2-(2-bromoethoxy)
ethane (53 lL, 0.422 mmol) were added. The reaction mixture was
heated at 90 °C under nitrogen for 3 h. After cooling to ambient
temperature, the mixture was poured into ice water (50 mL) and
extracted with CH2Cl2 (3 9 10 mL), washed with brine (15 mL)
and dried over MgSO4. Solvents were removed under reduced
pressure and the residue was purified over silica gel using hexane/
EtOAc (1 : 1) to give 8 (66 mg, 46%) as a white powder. 1H NMR
(400 MHz, CDCl3): d 9.88 (s, 1H), 8.64 (s, 1H), 8.16 (dd,
J = 8.1 Hz, 1.4 Hz, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.19 (dd,
J = 8.1 Hz, 1.4 Hz, 1H), 4.76 (t, J = 5.3 Hz, 2H), 3.97 (s, 3H),
3.85 (t, J = 5.3 Hz, 2H), 3.68 (t, J = 6.2 Hz, 2H), 3.35 (t,
J = 6.2 Hz, 2H), 2.18–2.08 (m, 9H), 1.78–1.67 (m, 6H). 13C
NMR (100 MHz, CDCl3): d 176.2, 150.9, 149.9, 125.2, 119.5,
114.3, 71.2, 70.4, 58.7, 56.4, 51.6, 41.8, 36.6, 29.9, 29.6. HRMS
calcd for C25H32BrN2O4 (M+H) 503.1540, found 503.1539.
bombardment was 90 min. For quality control, an aliquot of the
formulated solution was analyzed using the analytical HPLC system.
The identity of the 18F-labeled product was confirmed by comparison
with the HPLC retention time of the non-radioactive reference
compound RS-126 and by co-injection. Specific radioactivity of the
product was calculated by comparison of UV peak intensity with a
calibration curve of the non-radioactive reference compound.
In vitro binding assay
Membrane preparations obtained from CHO-K1 cells stably trans-
fected with human CB1 or CB2 (PerkinElmer, 0.5 lg/tube for hCB1
and hCB2) were incubated at 30 °C for 90 min with increasing
concentrations of test compounds (1 pM to 10 lM). [3H]CP-55,940
(1.4 nM; PerkinElmer) was used as radioligand, an agonist with Ki
values of 0.58 nM and 0.69 nM toward hCB1 and hCB2,
respectively. Non-specific binding was defined by the presence of
5 lM WIN-55212-2 (Biotrend AG; Ki hCB1 = 1.89, Ki
hCB2 = 0.28 nM). Inhibition constants Ki values were calculated
using the Cheng–Prusoff equation. KD values of 0.14 and 0.11 nM
from PerkinElmer were used for calculations for [3H]CP-55,940
binding to hCB1 and hCB2 receptors, respectively.
Synthesis of N-(1-adamantyl)-1-(2-(2-fluoroethoxy)ethyl)-8-
methoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide (RS-126). To
a
stirred solution of acetonitrile (1 mL), potassium fluoride
(9.23 mg, 0.159 mmol) and Kryptofix (59.8 mg, 0.159 mmol) were
added. The reaction mixture was heated to 90 °C, then compound 8
(20 mg, 0.040 mmol) was added and the mixture was stirred for
3 h. After cooling to ambient temperature, water (25 mL) was
added and the mixture was extracted with EtOAc (3 9 5 mL). The
combined organic layers were dried over MgSO4 and solvents were
removed under reduced pressure. The residue was purified over
silica gel using hexane/EtOAc (3 : 2 to 1 : 2) to give RS-126
(4 mg, 23%) as a yellowish powder. 1H NMR (400 MHz, CDCl3): d
9.90 (s, 1H), 8.66 (s, 1H), 8.17 (dd, J = 8.1 Hz, 1.4 Hz, 1H), 7.39
(t, J = 8.1 Hz, 1H), 7.19 (d, J = 7.7 Hz, 1H), 4.77 (t, J = 4.9 Hz,
2H), 4.54–4.40 (m, 2H), 3.97 (s, 3H), 3.88 (t, J = 4.9 Hz, 2H),
3.68–3.59 (m, 2H), 2.22–2.08 (m, 9H), 1.78–1.67 (m, 6H). 13C
NMR (100 MHz, CDCl3): d 178.7, 150.9, 84.1, 82.5, 70.3, 65.5,
56.8, 56.4, 52.4, 41.7, 36.5, 29.5, 25.0, 17.0. HRMS calcd for
C25H32FN2O4 (M+H) 443.2341, found 443.2340.
For the murine CB2 binding affinity, an adapted protocol using
membrane preparations from CHO cells expressing murine CB2
receptors in conjunction with [3H]CP-55,940 was used. Binding was
performed in 0.2 mL buffer pH 7.4 containing 50 mM tris(hydrox-
ymethyl)aminomethane (Tris), 5 mM MgCl2, 2.5 mM ethylene
glycol tetraacetic acid (EGTA) and 0.1% fatty acid free bovine
serum albumin (BSA) for 1 h at 30 °C under light shaking. The
reaction was terminated by rapid filtration through microfiltration
plates coated with 0.5% polyethylenimine (UniFilter GF/B filter
plate; Packard, Perkin Elmer, Wokingham, UK). Bound radioactiv-
ity was analyzed for Ki using nonlinear regression analysis (Activity
Base; ID Business Solution, Limited, Guilford, UK).
Animals
Animal care and experiments were conducted in accordance with
Swiss Animal Welfare legislation and were approved by the
Veterinary Office of Canton Zurich, Switzerland. Five-weeks old
male Wistar (Crl:WI) rats and 5-weeks old male CD1 (Crl:CD1)
mice were supplied by Charles River (Sulzfeld, Germany).
Radiochemistry
[
18F]-fluoride was produced via the 18O(p,n)18F nuclear reaction by
bombardment of 98% enriched 18O-water using a Cyclone 18/9
cyclotron (18-MeV; IBA, Belgium). Aqueous 18Fꢀ was trapped on a
hydrophilic anion exchange cartridge (Waters SepPak Accell QMA
cartridge carbonate) and eluted with a tetrabutylammoniumhydrox-
ide solution (TBAOH, 0.2 M in MeOH, 1 mL) into a reaction vessel.
The solvents were evaporated at 90 °C under reduced pressure with a
gentle inflow of nitrogen gas. After addition of acetonitrile (1 mL),
azeotropic drying was carried out. This procedure was repeated twice
to afford dry [18F]TBAF complex. A solution of precursor compound
8 (1 mg, 2 lmol) in DMF (0.3 mL) was added and the reaction
mixture was stirred at 110 °C for 10 min. After dilution with water
(2.7 mL), the crude product was purified by semi-preparative HPLC
system. The collected product fraction was diluted with water
(10 mL), trapped on a C18 cartridge (Waters, preconditioned with
5 mL EtOH and 10 mL water), washed with water (5 mL), and
eluted with EtOH (0.5 mL) through a sterile filter (0.2 lm). The
volume of EtOH was decreased under reduced pressure until ca.
0.1 mL and water (2 mL) was added to give a final EtOH
concentration of 5%. The total synthesis time from end of
In vitro stability studies
The stability of [18F]RS-126 was investigated in rat and human
plasma and in rat and mouse brain homogenates at 37 °C at various
incubation times (0–120 min). Phosphate-buffered saline (PBS,
0.15 M, pH 7.4) was used as a control.
For plasma, [18F]RS-126 formulated solution (10 lL, 3 MBq)
was added to PBS (300 lL), rat (300 lL), and human plasma
(300 lL), respectively. The mixtures were vortexed and incubated
at 37 °C under shaking. An aliquot of 100 lL from each sample
was taken at different time points (30, 60 and 120 min) and mixed
with 100 lL ice-cooled acetonitrile and centrifuged (3 min,
4500 g). The supernatants were analyzed by radio-TLC Instant
Imager (Packard, Canberra Company, Meriden, Connecticut, USA)
using hexane/EtOAc (1 : 4) as the eluent.
For the in vitro stability studies of [18F]RS-126 in the brain of a rat
and mouse, whole brains were removed after decapitation and stored
at ꢀ80 °C until use. For the actual experiment, the brains were thawed
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716

6
R. Slavik et al.
and homogenized in PBS (3 mL; GIBCO Lucerne, Switzerland) with
Ex vivo biodistribution studies
For biodistribution studies, a formulated solution of [18F]RS-126
(6–15 MBq, 0.06–0.16 nmol) was administered via tail vein
injection into Wistar rats (n = 3, 316–355 g). For blocking condi-
tions, GW405833 (1.5 mg/kg) was injected intravenous shortly
before radiotracer injection (n = 3, 330–356 g). For in vivo appli-
cations, 1.5 mg GW405833 was dissolved in 1 mL solution
containing 10% ethanol, 2% cremophor, and 88% water for
injection. Animals were euthanzised under anesthesia with isoflu-
rane by decapitation at 10 min p.i. Blood and tissues were collected,
weighed, and radioactivity measured in a gamma-counter (Wizard;
PerkinElmer). The accumulated radioactivity in the tissues was
expressed as percentage of injected dose per gram wet tissue
normalized to 1 kg body weight of the animals (norm. %ID/g
tissue).
a polytron on ice. The homogenates (400 lL) were incubated with
[
18F]RS-126 (15 lL, 7 MBq) at 37 °C under light shaking. An
aliquot (100 lL) was taken at 15, 30, 60 and 120 min and mixed with
ice-cooled acetonitrile (100 lL). The samples were vortexed, cen-
trifuged (5 min, 4500 g) and the supernatants analyzed by radio-
HPLC and radio-TLC using hexane/EtOAc (1 : 4) as the eluent.
In vitro autoradiography
Rat spleen tissue embedded in TissueTek was cut into 20 lm thick
sections on a cryostat (Cryo-Star HM 560 MV; Microm, Thermo
Scientific, Wilmington, DE, USA). The slices were absorbed on
SuperFrost Plus slides (Menzel, Braunschweig, Germany) and stored
at ꢀ80 °C until use. For the experiment, the slices were thawed on
ice and preconditioned at 0 °C in buffer (pH 7.4) containing 50 mM
TRIS/HCl and 5% BSA (incubation buffer). The tissue slices were
dried and then incubated with 600 lL incubation buffer containing
In vivo rat spleen-PET imaging
[
18F]RS-126 (0.6 nM) for 15 min at 21°C in a humid chamber.
For rat spleen-PET scans, animals (n = 9, 260–400 g) were
anesthetized with isoflurane and 10–17 MBq (0.3–0.5 nmol)
Under blockade conditions, AM251 (5 lM, CB1 inverse agonist,
low CB2 binding affinity, Ki = 2.3 lM) or GW405833 (5 lM, CB2
inverse agonist, high CB2 binding affinity, Ki = 3.9 nM) was added
to the incubation buffer containing the radioligand. The slices were
washed with washing buffer (50 mM TRIS/HCl, 1% BSA, 5%
EtOH, pH 7.4) for 2 min (29) and with distilled water for 5 s (29)
on ice. After drying, the slices were exposed (30 min) to appropriate
phosphor imager plates (Fuji, Dielsdorf, Switzerland) and the films
were scanned in a BAS5000 reader (Fuji).
[
18F]RS-126 was injected via the tail vein. Depth of anesthesia
was monitored by measuring respiratory frequency (SA Instru-
ments, Inc., Stony Brook, NY, USA). Body temperature was
controlled by a rectal probe and kept at 37 °C by a thermocoupler
and
a heated air stream. Rats under baseline (n = 5) and
displacement (n = 4) conditions were scanned for 90 min in
dynamic PET acquisition mode. Rats in the displacement exper-
iment additionally received 1.5 mg/kg GW405833 intravenously
10 min after radiotracer injection respectively to the start of PET
acquisition. PET data were reconstructed in user-defined time
frames with a voxel size of 0.3875 9 0.3875 9 0.775 mm3 by
two-dimensional-ordered subsets expectation maximization. Ran-
dom and single but no attenuation correction was applied. PET
acquisitions were followed by a CT for anatomical orientation.
Image files were analyzed with PMOD 3.6 software (PMOD
Technologies Ltd., Zurich, Switzerland). Spleen and muscle
regions were defined manually and tissue radioactivity was
expressed as standardized uptake values (SUVs), that is, the
decay-corrected radioactivity per cm3 divided by the injected
radioactivity dose per gram of body weight.
Determination of LogD7.4
The shake-flask method was used to determine the partition
coefficient D. In brief, n-octanol saturated with phosphate buffer
pH 7.4 (0.5 mL) and phosphate buffer saturated with n-octanol
(0.5 mL) were mixed with [18F]RS-126 (20 lL). The samples were
shaken for 15 min and centrifuged at 5000 g for 5 min at 21°C.
Layers were separated and radioactivity in each layer was measured
in a gamma counter (Wizard; PerkinElmer). LogD is expressed as
the logarithm of the ratio between the radioactivity concentrations
(Bq/mL) of the octanol and the buffer phase.
Ex vivo metabolic stability
In vivo mouse brain-PET imaging after lipopolysaccharide-induced
neuroinflammation
The radiotracer was administered intravenously to Wistar rats
(n = 2, 251 g and 330 g, 100 and 124 MBq, respectively, 1–
4 nmol) by tail vein injection. From the first rat (251 g), blood
samples were withdrawn from the tail artery at 15, 30 and 45 min
p.i.. The rat was killed by decapitation at 60 min p.i. to collect
blood, spleen, and brain. Blood from the second rat (330 g) was
collected from the tail artery at 5 min p.i.. The rat was killed 15 min
p.i. to collect blood, spleen, and brain. All the blood samples were
collected in heparin-coated tubes (BD Vacutainers, Allschwil,
Switzterland) and centrifuged at 5000 g for 5 min at 4 °C. The
proteins of the plasma were precipitated by addition of an equal
volume of ice-cooled acetonitrile and centrifuged at 5000 g for
5 min. The spleen and brain tissues were homogenized in PBS
(2 mL) for 2 min with a polytron. Ice-cooled acetonitrile (2 mL)
was added to precipitate proteins followed by centrifugation at
5000 g for 5 min. Supernatants were analyzed by radio-TLC using
hexane/EtOAc (1 : 4). Results are expressed as percentage of the
radioactivity of intact parent compound from total radioactivity.
Male CD1 mice (28–36 g) were intraperitoneally injected with
10 mg/kg lipopolysaccharide (LPS; E. coli strain O111:B4, Sigma
L4130; Sigma, St Louis, MO, USA, dissolved in 0.9% NaCl,
n = 11) or vehicle (0.9% NaCl, n = 6) 5 or 7 days prior brain-PET
imaging. For blocking experiments, LPS-treated mice received
2 mg/kg GW405833 subcutaneously 30 min before radiotracer
administration (n = 5). All other mice (n = 12) received an injection
of the vehicle solution (10% ethanol, 2% Cremophor, 88% water for
injection). Mice were intravenously administered with 3–11 MBq
(0.1–0.3 nmol)
[
18F]RS-126 and PET acquisition was started
simultaneous with injection. Dynamic PET scans lasted for
60 min and were followed by CT acquisition. Mouse monitoring
and PET data reconstruction was performed as described above.
Time activity curves (TACs) of the brain regions, defined on the
mouse MRI T2 template (PMOD Technologies Ltd.), were calcu-
lated and expressed as SUVs.
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716

PET radiotracer for imaging CB2 receptor
7
Statistics
desired fluorinated analog RS-030 in an overall yield of
30%.
For each group, the mean value was calculated and expressed with
standard deviation or standard error. Differences in mean values
were evaluated by an unpaired two-tailed student’s t-test (GraphPad
Prism 6.0 software; GraphPad Software Inc., San Diego, CA, USA).
A p-value < 0.05 was considered significant.
Compound 5 in Scheme 2 was obtained after demethyla-
tion of RS-016 using lithium chloride. The introduction of
the fluoroethyl side chain was achieved by O-alkylation of
compound 5 with fluoroethyl tosylate to yield the desired
fluoroethoxy derivative RS-122 in 79% yield.
Saponification of compound
2 with 10% NaOH
Results
(Scheme 3) yielded carboxylic acid 6, which was subse-
quently coupled with 1-aminoadamantane to afford com-
Chemistry
Based on the core structure of RS-016, which was previously
labeled in our group with carbon-11 (Slavik et al. 2015b),
three novel fluorinated analogs of RS-016 were designed
(Fig. 1) and synthesized.
pound 7. N-alkylation of compound
7 using bis(2-
bromoethyl)ether gave compound 8 in 46% yield, which
was then fluorinated with KF-Kryptofix to yield the
fluoroethyl ether derivative RS-126 in 23% yield.
Compound RS-030 was synthesized following a similar
synthetic procedure described for RS-016 (Slavik et al.
2015b) starting from commercially available anisidine and
diethyl 2-(ethoxymethylene)malonate as shown in
Scheme 1. Compound 2 was prepared via the Gould–
Jacobs reaction at high temperatures. N-alkylation under
basic conditions afforded compound 3 in 75% yields.
Quinoline acid 4 was obtained by saponification of the
ethyl ester 3 with 10% sodium hydroxide. Amide bond
formation using HBTU as the coupling reagent provided
compound RS-028 in a good yield (84%). The alcohol was
subsequently reacted with DAST according to a previously
reported patent (Jasys and Volkmann 2000) to afford the
In vitro binding affinity studies
The three novel fluorinated analogs of RS-016 were inves-
tigated for their binding affinities toward CB2 and selectivity
over CB1. Competitive binding experiments were carried out
with each candidate compound using [3H]CP55940, a well-
characterized CB1/CB2 agonist, and employing membranes
prepared from cells expressing human CB1 or CB2 recep-
tors. Non-radioactive WIN-55212-2 was used for the deter-
mination of non-specific binding. The displacement curves
showing the averaged values of three IC50 measurements are
depicted in Figure S1. The inhibition constants (Ki) of all the
Scheme 1 Synthesis of 3-fluoroadamantyl derivative RS-030. DAST, N,N-diethylaminosulfur trifluoride; HBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate.
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716

8
R. Slavik et al.
Scheme 2 Synthesis of fluoroethoxy derivative RS-122.
Scheme 3 Synthesis of fluoroethyl ether derivative RS-126.
tested compounds RS-030, RS-122, and RS-126 (Table 1)
were estimated using the Cheng–Prusoff equation.
fluorinated crude product was purified by semi-HPLC. In a
typical experiment, a moderate radiochemical yield of ~ 6%
(decay corrected) was achieved with a radiochemical purity
> 99%. The average specific activity for [18F]RS-126 was
98 GBq/lmol at the end of the radiosynthesis. The identity
of the radiolabeled compound was confirmed by co-injection
with the non-radioactive reference compound RS-126.
The replacement of the adamantyl group with the fluori-
nated adamantyl moiety yielded RS-030 with a favorable Ki
value of 2.1 nM toward CB2. Substitution of the methoxy
group in RS-016 with fluoroethyl resulted in RS-122, which
showed a 100-fold loss in affinity toward CB2 (Ki 72 nM).
The 2-fluoroethyl derivative RS-126 (Ki 1.2 nM) represents
the most potent CB2 ligand of all the three ligands
investigated, indicating that bioisosteric replacement of a
proton with a fluorine atom in the ethyl side chain does not
affect CB2 affinity. This potent CB2 ligand exhibited
> 8000-fold (Ki CB1 > 10 lM) selectivity over CB1. In a
further experiment using membranes expressing mouse CB2,
a Ki value of 4.5 nM was obtained for RS-126. This result is
comparable to the human CB2 Ki value of 1.2 nM, indicating
no significant species differences.
In vitro characterization of [18F]RS-126
The shake-flask method was applied to measure the distri-
bution of [18F]RS-126 in n-octanol/phosphate buffer (pH
7.4). The obtained logD7.4 value of 1.99 ꢁ 0.02 (n = 5)
suggests that [18F]RS-126 is sufficiently lipophilic for free
diffusion across the blood–brain barrier (Dishino et al.
1983).
The radiotracer was chemically stable for up to 2 h (data
not shown). Plasma stability tests were carried out in rat and
human plasma over a period of 120 min at 37 °C. No
radioactive degradation or biotransformation products of
[18F]RS-126 were detected in human or rat plasma after
120 min incubation time.
Radiosynthesis
[18F]RS-126 was prepared via nucleophilic substitution of
the bromo precursor 8 with [18F]TBAF (Scheme 4) and the
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716

PET radiotracer for imaging CB2 receptor
9
Table 1 Binding affinity (Ki) values of known cannabinoid ligands and
novel fluorinated derivatives with RS-016 as lead structure.
detected, which did not permit data evaluation. In the above
section, we already proved that in vitro incubation of [18F]
RS-126 with rat and mouse brain homogenates emerged no
biotransformation of the parent radioligand.
Compound
Ki CB1 [nM] Ki CB2 [nM] References
AM251
7.49
2290
Lan et al. 1999
In vivo PET imaging of peripheral CB2 in rat spleen with
[18F]RS-126
WIN-55212-2 1.89
0.28
Showalter et al. 1996
Showalter et al. 1996
Valenzano et al. 2005
Slavik et al. 2015b
This work (n = 3)
This work (n = 3)
This work (n = 3)
CP-55,940
GW405833
RS-016
0.58
0.69
In vivo accumulation of [18F]RS-126 in the CB2-positive rat
spleen was investigated by PET imaging (Fig. 3). The spleen
was clearly visible during the first 19 min after [18F]RS-126
injection (baseline). For displacement studies, the time point
for injection of the CB2-selective agonist GW405833 was
chosen at 10 min post radiotracer injection because at that
point, spleen-radioactivity was still high enough to be
displaced and was markedly discriminated from the back-
ground. The radioactivity in the spleen decreased more
rapidly after [18F]RS-126 displacement and visible in the
second time frame (10–19 min). The specific spleen-binding
of [18F]RS-126 which was measured within the first 9 min
was successfully displaced after administration of
GW405833. The corresponding TACs from spleen and
muscle of baseline and displacement experiments are shown
in Figure S2. Clearance of [18F]RS-126 from spleen tissue
was considerably enhanced after administration of the
displacer. The spleen-TAC reached background levels
approx. 5 min after displacer injection.
4772
3.90
> 10 000
> 10 000
> 10 000
> 10 000
0.7 ꢁ 0.6
2.1 ꢁ 1.5
72 ꢁ 102
1.2 ꢁ 0.8
RS-030
RS-122
RS-126
Brain homogenates from rat and mouse were incubated
with [18F]RS-126 at 37 °C under light shaking. After
120 min of incubation time, 97% and 98% of the total
radioactivity was assigned to the intact parent compound in
the rat and mouse brain samples, respectively.
Autoradiography images showed high radioactivity accu-
mulation of [18F]RS-126 to rat spleen tissue, an organ with
high basal levels of CB2 receptors, under baseline conditions
(Fig. 2). Blocking experiments using rat spleen slices that
were co-incubated with [18F]RS-126 and the CB2 inverse
agonist GW405833 (5 lM, Ki toward CB2 = 3.9 nM, Ki
toward CB1 = 4772 nM (Valenzano et al. 2005)) displayed
a substantially reduced radioactive signal. Co-incubation of
[18F]RS-126 with an excess amount of the CB1 inverse
agonist AM251 (5 lM, Ki toward CB2 = 2.3 lM (Lan et al.
1999)) using rat spleen slices showed similar intensity of
radioactive signal as under baseline conditions, indicating
selectivity of [18F]RS-126 for CB2 over CB1.
Ex vivo biodistribution of [18F]RS-126 in rats
Figure 4 shows the biodistribution data of [18F]RS-126 in
rats at 10 min post injection. The highest accumulation of
radioactivity was found in the liver, spleen, and small
intestine followed by adrenal glands and kidneys. The low
uptake of radioactivity in the bone suggests that no
significant defluorination has occurred during the experi-
ment. To assess the specificity of [18F]RS-126 uptake in
CB2-positive organs such as the spleen, the CB2 receptor
specific ligand GW405833 was utilized as a blocking agent.
As shown in Fig. 4, a significant reduction of radioactivity
uptake by 79% (p = 0.008) was observed for [18F]RS-126 in
the spleen tissue, indicating high specific binding of [18F]RS-
126 to peripheral CB2. Besides the spleen, a significant
reduction of radioactivity was found in the blood and lung
after GW405833 administration. It has been reported that
lung (Brown et al. 2002) and blood cells (Galiegue et al.
Ex vivo metabolic stability of [18F]RS-126 in rats
The ex vivo metabolic fate of [18F]RS-126 was studied using
healthy rat blood plasma, spleen, and brain. In spleen
samples, 81% and 75% of the total radioactivity were
assigned to the intact parent compound at 15 and 60 min p.i.,
respectively. In blood plasma samples, only one
radiometabolite, which was more hydrophilic than [18F]RS-
126, was detected. The percentage of intact compound in
blood plasma decreased to 82% at 5 min p.i., 55% at 10 min
p.i., 33% at 30 min p.i., 31% at 45 min p.i. and 23% at
60 min p.i.. In brain samples, negligible radioactivity was
Scheme 4 Radiosynthetic scheme for the
preparation of [18F]RS-126. TBAF, tetra-n-
butylammonium fluoride.
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716

10
R. Slavik et al.
1995) do express CB2, however, at much lower levels than
spleen. The high standard deviation of the small intestine in
blockade group is addressed to one animal (animal no. 1 in
Table S1) with very high radiotracer uptake for which we
have no explanation so far. Absolute values for each animal
are listed in Table S1.
In vivo brain-PET of CB2 in a mouse model of
neuroinflammation using [18F]RS-126
In our previous studies, a LPS-induced neuroinflammation
mouse model was used to study radiotracer uptake and binding
to the brain (Slavik et al. 2015a,b). CB2-mRNA levels
increased in the mouse brain 5 days after LPS treatment
(Slavik et al. 2015b). In this work, mice were injected with
10 mg/kg LPS 5 or 7 days prior [18F]RS-126 PET imaging
(Fig. 5). Control animals were administered with 0.9% NaCl
(vehicle). However, compared to control animals, we did not
find an increased accumulation of [18F]RS-126 in the brains of
LPS-treated mice neither at 5 nor 7 days post treatment.
Additionally, 2 mg/kg GW405833 was subcutaneously
injected 30 min prior [18F]RS-126 administration. Brain-
TACs of this group showed the same course as the other two
groups. TACs of individual brain regions (e.g. cortex, striatum,
hippocampus cerebellum etc.) were analyzed, however, with a
similar result as shown for the whole brain (data not shown).
Defluorination was observed 30 min after radiotracer injec-
tion, which led to incorporation of radioactivity in the cranial
bone and increased SUV in whole brain-TACs (Fig. 5)
because of spillover of radioactivity.
Fig. 2 Representative in vitro autoradiograms of rat spleen tissues
after incubation with [18F]RS-126 under baseline and blocking condi-
tions (5 lM cannabinoid receptor type 1 (CB1) agonist AM251 or 5 lM
CB2 agonist GW405833).
Discussion
RS-016, which was previously prepared in our laboratory,
exhibits high binding affinity toward CB2 (Ki = 0.7 nM),
good selectivity over CB1 (Ki > 10 lM), and an appropriate
lipophilicity (logD7.4 = 2.78) for brain penetration (Slavik
et al. 2015b). Unfortunately, its structure only allows for
radiolabeling with carbon-11, an isotope with a relatively
short physical half-life of 20.3 min. In this study, three novel
fluorinated analogs of RS-016 were designed and synthe-
sized. The 3-fluoroadamantyl derivative RS-030 was pre-
pared from the 3-hydroxyadamantyl intermediate RS-028
using DAST as the fluorinating agent. RS-122 was obtained
in 79% yield by treatment of the cesium salt of the phenolic
precursor 5 with fluoroethyl tosylate. The fluoroethyl ether
derivative RS-126 was obtained by reacting the bromo
precursor 8 with potassium fluoride and kryptofix. The yield
of this reaction was relatively low (23%) probably because of
a competing elimination reaction. Since sufficient amount of
RS-126 was obtained for in vitro evaluation, no efforts were
undertaken to optimize the chemical yield. From all the
fluorinated analogs of RS-016 synthesized, compound RS-
126 exhibited the highest binding affinity (Ki = 1.2 nM) to
CB2 and was therefore selected for further evaluation.
Fig. 3 Representative coronal positron emission tomography (PET)
images showing the abdominal region of two rats after [18F]RS-126
injection under baseline and displacement (1.5 mg/kg cannabinoid
receptor type 2 (CB2)-selective agonist GW405833 injected 10 min
after radiotracer) conditions. PET images were averaged to three time
frames as indicated. White arrowhead indicates the spleen. Injection of
GW405833 at 10 min displaced [18F]RS-126 from spleen tissue, which
is no more visible in the last two time frames (right column). St,
stomach; Li, liver; Int, intestine. Color bar indicates standardized
uptake values (SUV).
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716

PET radiotracer for imaging CB2 receptor
11
Fig. 4 Radioactivity accumulation of [18F]
RS-126 in selected rat tissue 10 min post
injection under baseline (black bars) and
blocking (gray bars; pre-administration of
1.5 mg/kg GW405833) conditions. Data are
presented as the mean of the percentage of
injected dose per gram tissue normalized to
the
body
weight
(kg)
of
the
animals ꢁ standard error [% norm. ID/g
tissue, means ꢁ standard error (n = 3)].
Absolute values are listed in Table S1.
Fig. 5 Whole brain [18F]RS-126 time activity
curves in a LPS-induce neuroinflammatory
mouse model. Mice were intraperitoneal
administered with 10 mg/kg LPS or vehicle
(0.9% NaCl). [18F]RS-126 PET imaging was
performed at 5 or 7 days post treatment.
Mice were further injected with 2 mg/kg
GW405833 or the corresponding vehicle
subcutaneous 30 min prior PET acquisition.
Note: Increasing SUV values from 30 min
onwards are attributed to defluorination of
[
18F]RS-126 followed by accumulation in the
cranial bone and spill over to the brain.
For the radiolabeling of RS-126 with fluorine-18, we
initially used K2.2.2/K[18F]F complex as the fluorinating
reagent, but very low radiochemical yields (< 1%) were
obtained under various radiolabeling conditions. Increasing
the reaction temperature as well as varying the reaction
solvent and reaction time using K2.2.2/K[18F]F complex as
the fluorinating reagent did not significantly improve the
radiochemical yield. Treatment of the bromide precursor 8
with [18F]TBAF as the radiofluorination reagent in the
presence of TBAOH as the base afforded target compound
[18F]RS-126 in 6% radiochemical yield. Replacing the more
basic TBAOH by TBAHCO3 with the aim to reduce
potential elimination products also did not lead to any
improvement in the radiochemical yield of [18F]RS-126. In
order to improve the radiochemical yield of [18F]RS-126, we
suggest to use a tosylate or mesylate precursor in future
studies. A higher 18F-incorporation is expected because of
the fact that tosyl or mesyl is a better leaving group compared
to bromide.
The lipophilicity of [18F]RS-126 was determined using the
shake-flask method and gave a logD7.4 value of 1.99, which is
lower than the logD7.4 value of [11C]RS-016 (2.78). This value
is still within the optimal lipophilicity range of 1–3 for brain-
penetrating compounds. Autoradiographic studies with [18F]
RS-126 showed high specific binding to CB2-positive rat
spleen tissue sections. Compared to [11C]RS-016, similar
in vitro/ex vivo metabolite stabilities were observed for [18F]
RS-126. No degradation product was detected in in vitro
assays with rat or human plasma. In vivo, rat spleen samples
revealed 81% of the intact parent compound at 15 min post
injection, which is similar to the value obtained for [11C]RS-
016 at 20 min p.i.. In vivo displacement studies confirmed
selective binding of [18F]RS-126 to CB2 in the rat spleen.
Biodistribution studies with rats revealed 79% specific binding
of [18F]RS-126 to spleen tissue. Based on the results we
previously obtained with [11C]RS-016 in LPS-treated mice, we
anticipated similar results with [18F]RS-126. However, con-
trary to our expectations, we could not detect CB2 expression
© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 10.1111/jnc.13716

12
R. Slavik et al.
Figure S2. [18F]RS-126 time activity curves of rat spleen (circle)
and muscle (triangle) under baseline (n = 5) and displacement
(1.5 mg/kg CB2-selective agonist GW405833, n = 4) conditions.
Table S1. [18F]RS-126 biodistribution values in rat tissue at 10
min p.i. under baseline (n = 3) and blocking (n = 3; pre-adminis-
tration of 1.5 mg/kg GW405833).
in the brains of LPS-treated mice. Considering the cold mass
injected to LPS-treated mice which was based on specific
radioactivity calculations, a 5–7.5-fold higher RS-126 (0.1–
0.3 nmol) was injected to LPS-treated mice compared to RS-
016 (0.02–0.04 nmol). In addition, we found a lower gene
expression level of CB2 in LPS-treated mice of 6.5-fold
compared to 9.5-fold from our previous study with [11C]RS-
016. We thus speculate that the lower specific activity of [18F]
RS-126 (98 GBq/lmol compared to [11C]RS-016 with
545 GBq/lmol) hampers the detection by PET of the per se
moderate CB2 levels in the brains of LPS-treated mice.
Another drawback is the rapid in vivo metabolism of [18F]RS-
126 in mice which resulted in the formation of [18F]fluoride.
Based on these results, we cannot definitely conclude that [18F]
RS-126 crosses the blood–brain barrier. However, we assume
some permeation of [18F]RS-126 into the brain since the
structurally related [11C]KD2 (Mu et al. 2013) showed
moderate brain uptake in in vitro transport experiments.
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also thank the Swiss ALS Foundation and the Vontobel Foundation
for partly financing this project. The authors have no conflict of
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All experiments were conducted in compliance with the ARRIVE
guidelines.
Supporting information
Additional Supporting Information may be found online in the
supporting information tab for this article:
Figure S1. Displacement curves for compounds RS-030, RS-
122, and RS-126 and compared with RS-016 using membranes
expressing human CB2 receptors.
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